qiaamp ffpe dna extraction kit  (Qiagen)


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    Name:
    QIAamp DNA FFPE Tissue Kit
    Description:
    For purification of genomic DNA from formalin fixed paraffin embedded tissues Kit contents Qiagen QIAamp DNA FFPE Tissue Kit 50 preps 20 to 100L Elution Volume Up to 8 Sections Sample Formalin Fixed Paraffin Embedded Tissue Sample Silica Technology Genomic DNA Mitochondrial DNA Purification Spin Column Format Ideal for Real time PCR STR Analysis LMD PCR For Purification of Genomic DNA From Formalin fixed Paraffin embedded Tissues Includes 50 QIAamp MinElute Columns Proteinase K Buffers 2mL Collection Tubes Benefits Rapid purification of high quality ready to use DNA Consistent high yields Complete removal of contaminants and inhibitor
    Catalog Number:
    56404
    Price:
    242
    Category:
    QIAamp DNA FFPE Tissue Kit
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    Structured Review

    Qiagen qiaamp ffpe dna extraction kit
    QIAamp DNA FFPE Tissue Kit
    For purification of genomic DNA from formalin fixed paraffin embedded tissues Kit contents Qiagen QIAamp DNA FFPE Tissue Kit 50 preps 20 to 100L Elution Volume Up to 8 Sections Sample Formalin Fixed Paraffin Embedded Tissue Sample Silica Technology Genomic DNA Mitochondrial DNA Purification Spin Column Format Ideal for Real time PCR STR Analysis LMD PCR For Purification of Genomic DNA From Formalin fixed Paraffin embedded Tissues Includes 50 QIAamp MinElute Columns Proteinase K Buffers 2mL Collection Tubes Benefits Rapid purification of high quality ready to use DNA Consistent high yields Complete removal of contaminants and inhibitor
    https://www.bioz.com/result/qiaamp ffpe dna extraction kit/product/Qiagen
    Average 99 stars, based on 2928 article reviews
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    qiaamp ffpe dna extraction kit - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA"

    Article Title: Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA

    Journal: NPJ genomic medicine

    doi: 10.1038/s41525-017-0030-7

    Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival FFPE tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution
    Figure Legend Snippet: Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival FFPE tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

    2) Product Images from "Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies"

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies

    Journal: BMC Medical Research Methodology

    doi: 10.1186/s12874-018-0628-1

    Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit
    Figure Legend Snippet: Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit
    Figure Legend Snippet: Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit

    Techniques Used: RNA Extraction, Formalin-fixed Paraffin-Embedded

    3) Product Images from "Analytic validation and real-time clinical application of an amplicon-based targeted gene panel for advanced cancer"

    Article Title: Analytic validation and real-time clinical application of an amplicon-based targeted gene panel for advanced cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20616

    Generation of sample mixes for analytic validation DNA from seven original samples was diluted to generate seven mixes. A. AN3CA and MFE-296 cell lines were mixed 1:1 to create Mix A, which was then mixed 1:1 with HCC827 to create Mix B and once again to create Mix C. B. Two frozen tumor samples and C. two FFPE tumor samples were combined 1:1 and 85:15 to generate mixes D and E (frozen) and F and G (FFPE).
    Figure Legend Snippet: Generation of sample mixes for analytic validation DNA from seven original samples was diluted to generate seven mixes. A. AN3CA and MFE-296 cell lines were mixed 1:1 to create Mix A, which was then mixed 1:1 with HCC827 to create Mix B and once again to create Mix C. B. Two frozen tumor samples and C. two FFPE tumor samples were combined 1:1 and 85:15 to generate mixes D and E (frozen) and F and G (FFPE).

    Techniques Used: Formalin-fixed Paraffin-Embedded

    4) Product Images from "Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies"

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies

    Journal: BMC Medical Research Methodology

    doi: 10.1186/s12874-018-0628-1

    Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit
    Figure Legend Snippet: Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit
    Figure Legend Snippet: Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit

    Techniques Used: RNA Extraction, Formalin-fixed Paraffin-Embedded

    5) Product Images from "Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies"

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies

    Journal: BMC Medical Research Methodology

    doi: 10.1186/s12874-018-0628-1

    Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit
    Figure Legend Snippet: Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit
    Figure Legend Snippet: Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit

    Techniques Used: RNA Extraction, Formalin-fixed Paraffin-Embedded

    6) Product Images from "Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples"

    Article Title: Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127146

    Depth of Sequencing correlates with DNA quality. (A) Sample preparation workflow. DNA was isolated from fresh frozen or FFPE CRC liver metastasis resection specimens with Qiagen Blood and Tissue or FFPE kit, respectively. Frozen samples then directly underwent sequencing library preparation, pooling of libraries, quality control and sequencing. FFPE samples were additionally tested for DNA quality by qPCR. Library quality was tested with Bioanalyzer. For samples with low amounts of correctly sized DNA amplicons (fragments at 310bp), new libraries were prepared with higher starting DNA concentrations and re-analyzed with Bioanalyzer. Samples with yet low amounts of DNA with correct size and highly fragmented DNA were excluded. (B) ΔCq-values of quality control PCR indicate poor sample quality. DNA concentration of fragments between 250bp and 450bp after library preparation was calculated with Agilent Bioanalyzer and plotted against ΔCq values of FFPE quality control PCR. (C) higher ΔCq-values correlate with lower mean depth of sequencing. (D) Coverage distribution of amplicons from all paired FFPE and frozen samples, normalized to total sample coverage. Frozen samples had a mean depth of 4,622, FFPE samples 1,852.
    Figure Legend Snippet: Depth of Sequencing correlates with DNA quality. (A) Sample preparation workflow. DNA was isolated from fresh frozen or FFPE CRC liver metastasis resection specimens with Qiagen Blood and Tissue or FFPE kit, respectively. Frozen samples then directly underwent sequencing library preparation, pooling of libraries, quality control and sequencing. FFPE samples were additionally tested for DNA quality by qPCR. Library quality was tested with Bioanalyzer. For samples with low amounts of correctly sized DNA amplicons (fragments at 310bp), new libraries were prepared with higher starting DNA concentrations and re-analyzed with Bioanalyzer. Samples with yet low amounts of DNA with correct size and highly fragmented DNA were excluded. (B) ΔCq-values of quality control PCR indicate poor sample quality. DNA concentration of fragments between 250bp and 450bp after library preparation was calculated with Agilent Bioanalyzer and plotted against ΔCq values of FFPE quality control PCR. (C) higher ΔCq-values correlate with lower mean depth of sequencing. (D) Coverage distribution of amplicons from all paired FFPE and frozen samples, normalized to total sample coverage. Frozen samples had a mean depth of 4,622, FFPE samples 1,852.

    Techniques Used: Sequencing, Sample Prep, Isolation, Formalin-fixed Paraffin-Embedded, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Concentration Assay

    7) Product Images from "Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors"

    Article Title: Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors

    Journal: Genome Medicine

    doi: 10.1186/gm453

    Workflow of FFPE sample preparation and selection . Eighty-two FFPE blocks [ 19 ] were stained for p16 of which eight samples were excluded from further analysis, showing mixed p16 staining. Eight samples were excluded after the LCM step, yielding insufficient amounts or quality of DNA and two further samples were excluded due to inconsistent or borderline results in repeat E6 qPCR measurements. In total, 22 confirmed HPV+ (p16+ and E6 qPCR+) and 34 HPV- (p16- and E6 qPCR-) samples were suitable for further analysis. Following age and gender matching, 20 HPV+ HNSCC samples (red) and 20 HPV- HNSCC samples (grey) were then selected for the final analysis (next-generation (NG) sequencing).
    Figure Legend Snippet: Workflow of FFPE sample preparation and selection . Eighty-two FFPE blocks [ 19 ] were stained for p16 of which eight samples were excluded from further analysis, showing mixed p16 staining. Eight samples were excluded after the LCM step, yielding insufficient amounts or quality of DNA and two further samples were excluded due to inconsistent or borderline results in repeat E6 qPCR measurements. In total, 22 confirmed HPV+ (p16+ and E6 qPCR+) and 34 HPV- (p16- and E6 qPCR-) samples were suitable for further analysis. Following age and gender matching, 20 HPV+ HNSCC samples (red) and 20 HPV- HNSCC samples (grey) were then selected for the final analysis (next-generation (NG) sequencing).

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sample Prep, Selection, Staining, Laser Capture Microdissection, Real-time Polymerase Chain Reaction, Sequencing

    8) Product Images from "The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum"

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-016-0223-4

    Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit
    Figure Legend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Formalin-fixed Paraffin-Embedded

    9) Product Images from "Critical Issues in Mycobiota Analysis"

    Article Title: Critical Issues in Mycobiota Analysis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00180

    DNA isolation from human FFPE skin samples and ITS PCR amplification influenced by beat beating. (A) Significant difference in overall DNA yield from FFPE skin samples ( n = 10) with and without bead beating ( ** p
    Figure Legend Snippet: DNA isolation from human FFPE skin samples and ITS PCR amplification influenced by beat beating. (A) Significant difference in overall DNA yield from FFPE skin samples ( n = 10) with and without bead beating ( ** p

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Amplification

    10) Product Images from "Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method"

    Article Title: Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-12-12

    Pyrograms of the LINE-1, MLH1 and MGMT methylation markers for different couples of frozen/FFPE DNA . Pyrograms of LINE-1 marker are those obtained for couple n° 10 ( A and B ) and for MLH1 and MGMT markers those for couples n°13 ( C and D ) and n°6 ( E and F ) respectively. Arrows indicate positions of internal controls of conversion, demonstrating no residual cytosines at the non-CpG sites. Gray areas indicate polymorphisms, between T/C, generated by bisulfite treatment. Level of methylation for a given CpG dinucleotide is reported above it (gray square).
    Figure Legend Snippet: Pyrograms of the LINE-1, MLH1 and MGMT methylation markers for different couples of frozen/FFPE DNA . Pyrograms of LINE-1 marker are those obtained for couple n° 10 ( A and B ) and for MLH1 and MGMT markers those for couples n°13 ( C and D ) and n°6 ( E and F ) respectively. Arrows indicate positions of internal controls of conversion, demonstrating no residual cytosines at the non-CpG sites. Gray areas indicate polymorphisms, between T/C, generated by bisulfite treatment. Level of methylation for a given CpG dinucleotide is reported above it (gray square).

    Techniques Used: Methylation, Formalin-fixed Paraffin-Embedded, Marker, Generated

    11) Product Images from "A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry"

    Article Title: A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043842

    Limit of detection of the Therascreen EGFR Mutation Test Kit in comparison with direct sequencing. Serial dilutions of DNA from EGFR mutant and wild-type FFPE tumours were used to compare the relative sensitivities of both methods. ( A ), the Therascreen EGFR Mutation Test Kit was able to detect an EGFR mutation when the DNA from the mutant tumour represented 1% of the total DNA in half of the analyzed tumours. Sample S08-3853, which harbours an exon 19 deletion, is shown as an example. ( B ), the Therascreen EGFR Mutation Test Kit was able to identify EGFR mutation in a 5% dilution of the total DNA in all the tumours analyzed. Sample S09-397, which harbours an exon 19 deletion, is shown as an example. ( C ), at least 30% mutant DNA was necessary in a background of wild-type DNA to detect EGFR mutations by direct sequencing in most of the analyzed tumours. Sample S08-3853 is shown as an example. Percentages indicate the proportion of DNA from a mutant tumour relative to DNA from a wild-type tumour. The ΔCt cut-off value to detect the presence of an EGFR exon 19 deletion is provided by the manufacturer. It is derived from cell lines and synthetic constructs.
    Figure Legend Snippet: Limit of detection of the Therascreen EGFR Mutation Test Kit in comparison with direct sequencing. Serial dilutions of DNA from EGFR mutant and wild-type FFPE tumours were used to compare the relative sensitivities of both methods. ( A ), the Therascreen EGFR Mutation Test Kit was able to detect an EGFR mutation when the DNA from the mutant tumour represented 1% of the total DNA in half of the analyzed tumours. Sample S08-3853, which harbours an exon 19 deletion, is shown as an example. ( B ), the Therascreen EGFR Mutation Test Kit was able to identify EGFR mutation in a 5% dilution of the total DNA in all the tumours analyzed. Sample S09-397, which harbours an exon 19 deletion, is shown as an example. ( C ), at least 30% mutant DNA was necessary in a background of wild-type DNA to detect EGFR mutations by direct sequencing in most of the analyzed tumours. Sample S08-3853 is shown as an example. Percentages indicate the proportion of DNA from a mutant tumour relative to DNA from a wild-type tumour. The ΔCt cut-off value to detect the presence of an EGFR exon 19 deletion is provided by the manufacturer. It is derived from cell lines and synthetic constructs.

    Techniques Used: Mutagenesis, Sequencing, Formalin-fixed Paraffin-Embedded, Derivative Assay, Construct

    12) Product Images from "Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling"

    Article Title: Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2015.268

    Flowchart of FFPE DNA sample and QC assays. After DNA extraction, three QC assays were performed to validate the quality of the DNA: (1) QC-PCR was used to estimate the level of fragmentation by comparing two PCR products amplified from FFPE DNA with the amplified PCR products from HapMap DNA (NA12878). According to the results of the QC-PCR, samples were classified as good, medium or poor. (2) DNA concentrations were measured using a PicoGreen assay. (3) All DNA samples were analyzed on a TapeStation to view the fragmentation profile of the DNA. Either the profiled was rated as ‘flat' indicating that DNA was highly degraded or not present, or the profile was rated as ‘peak' indicating that the DNA was degrade but had a peak when looking at the electropherogram. If a sample was rated poor, had a DNA concentration less than 1 ng/ μ l and a ‘flat' fragmentation profile, the DNA sample had failed QC. Only selected DNA samples failing QC were passed on to library preparation and sequencing, if there was a known variant in the family to search for.
    Figure Legend Snippet: Flowchart of FFPE DNA sample and QC assays. After DNA extraction, three QC assays were performed to validate the quality of the DNA: (1) QC-PCR was used to estimate the level of fragmentation by comparing two PCR products amplified from FFPE DNA with the amplified PCR products from HapMap DNA (NA12878). According to the results of the QC-PCR, samples were classified as good, medium or poor. (2) DNA concentrations were measured using a PicoGreen assay. (3) All DNA samples were analyzed on a TapeStation to view the fragmentation profile of the DNA. Either the profiled was rated as ‘flat' indicating that DNA was highly degraded or not present, or the profile was rated as ‘peak' indicating that the DNA was degrade but had a peak when looking at the electropherogram. If a sample was rated poor, had a DNA concentration less than 1 ng/ μ l and a ‘flat' fragmentation profile, the DNA sample had failed QC. Only selected DNA samples failing QC were passed on to library preparation and sequencing, if there was a known variant in the family to search for.

    Techniques Used: Formalin-fixed Paraffin-Embedded, DNA Extraction, Polymerase Chain Reaction, Amplification, Picogreen Assay, Concentration Assay, Sequencing, Variant Assay

    13) Product Images from "Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer"

    Article Title: Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer

    Journal: Cancer Genomics & Proteomics

    doi:

    Gel electrophoresis of CTNNB1 gene (190 bp) from DNA retrieved from already immunostained FFPE IHC slides. L, Gel ladder as a control; lanes 1 to 11, numbers' of assessed samples; IHC, immunohisochemistry, FFPE, formalin-fixed, paraffin-embedded
    Figure Legend Snippet: Gel electrophoresis of CTNNB1 gene (190 bp) from DNA retrieved from already immunostained FFPE IHC slides. L, Gel ladder as a control; lanes 1 to 11, numbers' of assessed samples; IHC, immunohisochemistry, FFPE, formalin-fixed, paraffin-embedded

    Techniques Used: Nucleic Acid Electrophoresis, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

    14) Product Images from "Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics"

    Article Title: Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics

    Journal: Virchows Archiv

    doi: 10.1007/s00428-017-2288-7

    Analysis of 15 FFPE cancer samples with commercial cancer panels. Centrally as well as locally extracted DNA of molecularly pre-characterized cancer samples was sequenced by commercial cancer panels (CHPv2 and TSACP) at five different sequencing sites. Mutations ascertained by conventional Sanger or pyro-sequencing and reproduced by NGS are listed in supplementary Table 2. a Analysis of five Colon cancer samples. b Analysis of five Breast cancer samples. c Analysis of five Lung cancer samples, (#1–#5, respectively). Variant allelic frequencies, detected at different partner sites, are illustrated by bars as indicated. Samples not analyzed are indicated by “X”; variants not detected are indicated by open circles “ ○ .” WT wild type; a, b, and c PGM™ sequencing sites; d and e MiSeq™ sequencing sites
    Figure Legend Snippet: Analysis of 15 FFPE cancer samples with commercial cancer panels. Centrally as well as locally extracted DNA of molecularly pre-characterized cancer samples was sequenced by commercial cancer panels (CHPv2 and TSACP) at five different sequencing sites. Mutations ascertained by conventional Sanger or pyro-sequencing and reproduced by NGS are listed in supplementary Table 2. a Analysis of five Colon cancer samples. b Analysis of five Breast cancer samples. c Analysis of five Lung cancer samples, (#1–#5, respectively). Variant allelic frequencies, detected at different partner sites, are illustrated by bars as indicated. Samples not analyzed are indicated by “X”; variants not detected are indicated by open circles “ ○ .” WT wild type; a, b, and c PGM™ sequencing sites; d and e MiSeq™ sequencing sites

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, Next-Generation Sequencing, Variant Assay

    Multicenter study design for targeted NGS. a A commercial gene panel (Cancer Hotspot panel 2, CHPv2, Thermo Fisher Scientific) was applied to DNA from the LoVo cell line at four distinct dilutions at three PGM™ sequencing sites (a, b, and c) to demonstrate exemplarily sensitivity. Bioinformatics was performed locally. b Genomic DNA from 15 molecularly pre-characterized tumor samples (five breast, five lung, and five colon cancer cases) was analyzed with commercially available and custom-designed cancer gene panels on PGM™ and MiSeq™ benchtop sequencers at seven sequencing sites (a, b, c, d, e, f, and g). FFPE tissue sections of the very same tumor samples were delivered to the sites a, b, d, and e for local microdissection, DNA Isolation, QC/quantification, and commercial panel sequencing. Partner site c did not receive tissue sections for local DNA extraction and applied only centrally extracted DNA to commercial (c*) and cancer-specific gene panel sequencing (c). Bioinformatics of commercial cancer panel-based data was performed individually whereas cancer-specific panel-based data were collected centrally and compiled
    Figure Legend Snippet: Multicenter study design for targeted NGS. a A commercial gene panel (Cancer Hotspot panel 2, CHPv2, Thermo Fisher Scientific) was applied to DNA from the LoVo cell line at four distinct dilutions at three PGM™ sequencing sites (a, b, and c) to demonstrate exemplarily sensitivity. Bioinformatics was performed locally. b Genomic DNA from 15 molecularly pre-characterized tumor samples (five breast, five lung, and five colon cancer cases) was analyzed with commercially available and custom-designed cancer gene panels on PGM™ and MiSeq™ benchtop sequencers at seven sequencing sites (a, b, c, d, e, f, and g). FFPE tissue sections of the very same tumor samples were delivered to the sites a, b, d, and e for local microdissection, DNA Isolation, QC/quantification, and commercial panel sequencing. Partner site c did not receive tissue sections for local DNA extraction and applied only centrally extracted DNA to commercial (c*) and cancer-specific gene panel sequencing (c). Bioinformatics of commercial cancer panel-based data was performed individually whereas cancer-specific panel-based data were collected centrally and compiled

    Techniques Used: Next-Generation Sequencing, Sequencing, Formalin-fixed Paraffin-Embedded, Laser Capture Microdissection, DNA Extraction

    15) Product Images from "Biomarker-driven trial in metastatic pancreas cancer: feasibility in a multicenter study of saracatinib, an oral Src inhibitor, in previously treated pancreatic cancer"

    Article Title: Biomarker-driven trial in metastatic pancreas cancer: feasibility in a multicenter study of saracatinib, an oral Src inhibitor, in previously treated pancreatic cancer

    Journal: Cancer Medicine

    doi: 10.1002/cam4.27

    (A) Representative figure of patients archival tumor or fresh liver biopsies analyzed for LRRC19 > IGFBP2 and (B) PIK3CA mutation (3′ UTR) of PH1715 (patient enrolled in the biomarker portion of the study).
    Figure Legend Snippet: (A) Representative figure of patients archival tumor or fresh liver biopsies analyzed for LRRC19 > IGFBP2 and (B) PIK3CA mutation (3′ UTR) of PH1715 (patient enrolled in the biomarker portion of the study).

    Techniques Used: Mutagenesis, Biomarker Assay

    Study design: in the unselected portion, 17 patients were enrolled in the study. To move on to enroll 34 patients at least three responses were required. Only two patients made it to the 6-month endpoint. Study was amended for a biomarker-driven study. Results from our preclinical study on 24 patient-derived pancreatic explants identified the KTSP classifier LRRC19 > IGFBP2 and PIK3CA mutant as markers of sensitivity. These markers were used to screen patients for the biomarker-driven study. One patient with a PIK3CA mutation was enrolled in the study.
    Figure Legend Snippet: Study design: in the unselected portion, 17 patients were enrolled in the study. To move on to enroll 34 patients at least three responses were required. Only two patients made it to the 6-month endpoint. Study was amended for a biomarker-driven study. Results from our preclinical study on 24 patient-derived pancreatic explants identified the KTSP classifier LRRC19 > IGFBP2 and PIK3CA mutant as markers of sensitivity. These markers were used to screen patients for the biomarker-driven study. One patient with a PIK3CA mutation was enrolled in the study.

    Techniques Used: Biomarker Assay, Derivative Assay, Mutagenesis

    16) Product Images from "Chronic inflammation initiates multiple forms of K-Ras-independent mouse pancreatic cancer in the absence of TP53"

    Article Title: Chronic inflammation initiates multiple forms of K-Ras-independent mouse pancreatic cancer in the absence of TP53

    Journal: Oncogene

    doi: 10.1038/onc.2016.461

    Ras-independent tumorigenesis was the predominant pathway of carcinogenesis associated with chronic inflammation in the absence of TP53. ( A ) Detection of K-ras mutation. (a) In control mice, only wild-type (WT) DNA (negative control) signal was detected in absence of LNA probe, whereas addition of LNA probe resulted in its complete suppression. (b) In K-Ras G12D expressing mice (LSL G12D /Cre) adding the LNA probe resulted in suppression of WT signal and detection of mutant K-ras as indicated by the difference in the melting temperature for WT DNA and mutant K-ras. (c) The results of gDNA analysis of microdissected tissue from PDAC areas of tumor showing detection of only WT but not mutant K-ras. ( B ) Representative Immunohistochemical images showing for mutant Ras showing positive staining in LSL-K-ras G12V / Cre and LSL-K-ras G12D /Cre tumors, and negative staining in tumors of TP53 −/− /COX-2/Cre mice (scale bar: 200 μm, magnification 20 ×). ( C ) Representative immunohistochemical images showing focal positive staining for phospho-ERK (pERK), in tumors (two representative images of TP53 −/− /COX-2/Cre and TP53 −/− /COX-2/IKK/Cre tumors) (scale bar: 200 μm, magnification 10 ×).
    Figure Legend Snippet: Ras-independent tumorigenesis was the predominant pathway of carcinogenesis associated with chronic inflammation in the absence of TP53. ( A ) Detection of K-ras mutation. (a) In control mice, only wild-type (WT) DNA (negative control) signal was detected in absence of LNA probe, whereas addition of LNA probe resulted in its complete suppression. (b) In K-Ras G12D expressing mice (LSL G12D /Cre) adding the LNA probe resulted in suppression of WT signal and detection of mutant K-ras as indicated by the difference in the melting temperature for WT DNA and mutant K-ras. (c) The results of gDNA analysis of microdissected tissue from PDAC areas of tumor showing detection of only WT but not mutant K-ras. ( B ) Representative Immunohistochemical images showing for mutant Ras showing positive staining in LSL-K-ras G12V / Cre and LSL-K-ras G12D /Cre tumors, and negative staining in tumors of TP53 −/− /COX-2/Cre mice (scale bar: 200 μm, magnification 20 ×). ( C ) Representative immunohistochemical images showing focal positive staining for phospho-ERK (pERK), in tumors (two representative images of TP53 −/− /COX-2/Cre and TP53 −/− /COX-2/IKK/Cre tumors) (scale bar: 200 μm, magnification 10 ×).

    Techniques Used: Mutagenesis, Mouse Assay, Negative Control, Expressing, Immunohistochemistry, Staining, Negative Staining

    17) Product Images from "Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics"

    Article Title: Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics

    Journal: Virchows Archiv

    doi: 10.1007/s00428-017-2288-7

    Disease-specific gene panel-based analysis of FFPE cancer samples. Centrally extracted DNA of molecularly pre-characterized cancer samples was sequenced using cancer-specific, custom-designed cancer panel at seven sequencing sites. The same cases as depicted in Fig. 3 are used. a Colon/Lung Ca panel-based analysis of five Colon cancer samples. b Custom-designed Breast cancer panel-based analysis of five Breast cancer samples. c Custom-designed Lung Cancer panel-based analysis of five Lung cancer samples (#1–#5, respectively). Mutations ascertained by conventional Sanger or pyro-sequencing and reproduced by NGS are listed in supplementary Table 2. Detected variant allelic frequencies of KRAS (Colon Ca), PIK3CA / PTEN (Breast Ca), and EGFR mutations (Lung Ca) are illustrated by bars in corresponding colors/patterns (a, b, and c represent PGM; d, e, f, and g represent MiSeq sequencing sites). WT wild type
    Figure Legend Snippet: Disease-specific gene panel-based analysis of FFPE cancer samples. Centrally extracted DNA of molecularly pre-characterized cancer samples was sequenced using cancer-specific, custom-designed cancer panel at seven sequencing sites. The same cases as depicted in Fig. 3 are used. a Colon/Lung Ca panel-based analysis of five Colon cancer samples. b Custom-designed Breast cancer panel-based analysis of five Breast cancer samples. c Custom-designed Lung Cancer panel-based analysis of five Lung cancer samples (#1–#5, respectively). Mutations ascertained by conventional Sanger or pyro-sequencing and reproduced by NGS are listed in supplementary Table 2. Detected variant allelic frequencies of KRAS (Colon Ca), PIK3CA / PTEN (Breast Ca), and EGFR mutations (Lung Ca) are illustrated by bars in corresponding colors/patterns (a, b, and c represent PGM; d, e, f, and g represent MiSeq sequencing sites). WT wild type

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, Next-Generation Sequencing, Variant Assay

    Analysis of 15 FFPE cancer samples with commercial cancer panels. Centrally as well as locally extracted DNA of molecularly pre-characterized cancer samples was sequenced by commercial cancer panels (CHPv2 and TSACP) at five different sequencing sites. Mutations ascertained by conventional Sanger or pyro-sequencing and reproduced by NGS are listed in supplementary Table 2. a Analysis of five Colon cancer samples. b Analysis of five Breast cancer samples. c Analysis of five Lung cancer samples, (#1–#5, respectively). Variant allelic frequencies, detected at different partner sites, are illustrated by bars as indicated. Samples not analyzed are indicated by “X”; variants not detected are indicated by open circles “ ○ .” WT wild type; a, b, and c PGM™ sequencing sites; d and e MiSeq™ sequencing sites
    Figure Legend Snippet: Analysis of 15 FFPE cancer samples with commercial cancer panels. Centrally as well as locally extracted DNA of molecularly pre-characterized cancer samples was sequenced by commercial cancer panels (CHPv2 and TSACP) at five different sequencing sites. Mutations ascertained by conventional Sanger or pyro-sequencing and reproduced by NGS are listed in supplementary Table 2. a Analysis of five Colon cancer samples. b Analysis of five Breast cancer samples. c Analysis of five Lung cancer samples, (#1–#5, respectively). Variant allelic frequencies, detected at different partner sites, are illustrated by bars as indicated. Samples not analyzed are indicated by “X”; variants not detected are indicated by open circles “ ○ .” WT wild type; a, b, and c PGM™ sequencing sites; d and e MiSeq™ sequencing sites

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, Next-Generation Sequencing, Variant Assay

    Multicenter study design for targeted NGS. a A commercial gene panel (Cancer Hotspot panel 2, CHPv2, Thermo Fisher Scientific) was applied to DNA from the LoVo cell line at four distinct dilutions at three PGM™ sequencing sites (a, b, and c) to demonstrate exemplarily sensitivity. Bioinformatics was performed locally. b Genomic DNA from 15 molecularly pre-characterized tumor samples (five breast, five lung, and five colon cancer cases) was analyzed with commercially available and custom-designed cancer gene panels on PGM™ and MiSeq™ benchtop sequencers at seven sequencing sites (a, b, c, d, e, f, and g). FFPE tissue sections of the very same tumor samples were delivered to the sites a, b, d, and e for local microdissection, DNA Isolation, QC/quantification, and commercial panel sequencing. Partner site c did not receive tissue sections for local DNA extraction and applied only centrally extracted DNA to commercial (c*) and cancer-specific gene panel sequencing (c). Bioinformatics of commercial cancer panel-based data was performed individually whereas cancer-specific panel-based data were collected centrally and compiled
    Figure Legend Snippet: Multicenter study design for targeted NGS. a A commercial gene panel (Cancer Hotspot panel 2, CHPv2, Thermo Fisher Scientific) was applied to DNA from the LoVo cell line at four distinct dilutions at three PGM™ sequencing sites (a, b, and c) to demonstrate exemplarily sensitivity. Bioinformatics was performed locally. b Genomic DNA from 15 molecularly pre-characterized tumor samples (five breast, five lung, and five colon cancer cases) was analyzed with commercially available and custom-designed cancer gene panels on PGM™ and MiSeq™ benchtop sequencers at seven sequencing sites (a, b, c, d, e, f, and g). FFPE tissue sections of the very same tumor samples were delivered to the sites a, b, d, and e for local microdissection, DNA Isolation, QC/quantification, and commercial panel sequencing. Partner site c did not receive tissue sections for local DNA extraction and applied only centrally extracted DNA to commercial (c*) and cancer-specific gene panel sequencing (c). Bioinformatics of commercial cancer panel-based data was performed individually whereas cancer-specific panel-based data were collected centrally and compiled

    Techniques Used: Next-Generation Sequencing, Sequencing, Formalin-fixed Paraffin-Embedded, Laser Capture Microdissection, DNA Extraction

    18) Product Images from "Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues"

    Article Title: Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203608

    Storage of PET blocks for 9 years at lower temperatures prevents from DNA degradation. Analysis of DNA integrity from FFPE (A) and PFPE (B) tissues of rat liver, kidney, spleen, lung and intestine on Agilent 4200 TapeStation system with genomic DNA Analysis ScreenTape assay. PET blocks were stored prior to DNA extraction for 108 months at 22°C, 4°C, and -20°C. DNA was extracted from 3x 10μm sections in triplicates.
    Figure Legend Snippet: Storage of PET blocks for 9 years at lower temperatures prevents from DNA degradation. Analysis of DNA integrity from FFPE (A) and PFPE (B) tissues of rat liver, kidney, spleen, lung and intestine on Agilent 4200 TapeStation system with genomic DNA Analysis ScreenTape assay. PET blocks were stored prior to DNA extraction for 108 months at 22°C, 4°C, and -20°C. DNA was extracted from 3x 10μm sections in triplicates.

    Techniques Used: Positron Emission Tomography, Formalin-fixed Paraffin-Embedded, DNA Extraction

    Improved qPCR-performance of DNA from PET blocks stored for 9 years at lower temperatures. FFPE and PFPE animal tissues were stored prior to DNA extraction for 108 months at different temperatures (22°C, 4°C, and -20°C). qPCR was performed with four different SYBR-Green qPCR assays of the rat ACTB gene with amplicon length of 271, 523, 650 and 747 base pairs (bp). Triplicate extractions from each PET block were amplified. DNA from cryo-preserved rat tissue was used as reference, shown as dashed line. Mean Ct values with standard deviation from triplicate extractions from each of five different tissues (liver, kidney, spleen, lung and intestine) are shown for each assay.
    Figure Legend Snippet: Improved qPCR-performance of DNA from PET blocks stored for 9 years at lower temperatures. FFPE and PFPE animal tissues were stored prior to DNA extraction for 108 months at different temperatures (22°C, 4°C, and -20°C). qPCR was performed with four different SYBR-Green qPCR assays of the rat ACTB gene with amplicon length of 271, 523, 650 and 747 base pairs (bp). Triplicate extractions from each PET block were amplified. DNA from cryo-preserved rat tissue was used as reference, shown as dashed line. Mean Ct values with standard deviation from triplicate extractions from each of five different tissues (liver, kidney, spleen, lung and intestine) are shown for each assay.

    Techniques Used: Real-time Polymerase Chain Reaction, Positron Emission Tomography, Formalin-fixed Paraffin-Embedded, DNA Extraction, SYBR Green Assay, Amplification, Blocking Assay, Standard Deviation

    19) Product Images from "CRE: a cost effective and rapid approach for PCR-mediated concatenation ofKRAS andEGFR exons"

    Article Title: CRE: a cost effective and rapid approach for PCR-mediated concatenation ofKRAS andEGFR exons

    Journal: F1000Research

    doi: 10.12688/f1000research.6663.2

    Multiplex PCR amplification and concatenation of KRAS and EGFR exons generates CRE product. Panel A . PCR amplification of KRAS and EGFR exons using NCI-H1975 genomic DNA: Lane 1, KRAS exon 2 (151 bp) amplified with OAD176 and OAD177; Lane 2, EGFR exon 18 (209 bp) amplified with OAD 178 and OAD 144; Lane 3, EGFR exon 19 (178 bp) amplified with OAD 145 and OAD 146; Lane 4, EGFR exon 20 (246 bp) amplified with OAD 147 and OAD 150; Lane 5, EGFR exon 21 (251 bp) amplified with OAD 151 and OAD 152; Lane 6, Multiplex PCR of KRAS exon 2 and EGFR exons 18-21 with cocktail of primers used in Lanes 1–5. Concatenated KRAS and EGFR (CRE) product of ~915 bp amplified with OAD 176 and OAD 152 using multiplex PCR product as template derived from NCI-H1975 genomic DNA (shown in Panel B , Lane 2); derived from fresh frozen primary tumor genomic DNA (shown in Panel C , Lane 2); using tumor genomic DNA extracted from FFPE block (shown in Panel D, Lane 2).
    Figure Legend Snippet: Multiplex PCR amplification and concatenation of KRAS and EGFR exons generates CRE product. Panel A . PCR amplification of KRAS and EGFR exons using NCI-H1975 genomic DNA: Lane 1, KRAS exon 2 (151 bp) amplified with OAD176 and OAD177; Lane 2, EGFR exon 18 (209 bp) amplified with OAD 178 and OAD 144; Lane 3, EGFR exon 19 (178 bp) amplified with OAD 145 and OAD 146; Lane 4, EGFR exon 20 (246 bp) amplified with OAD 147 and OAD 150; Lane 5, EGFR exon 21 (251 bp) amplified with OAD 151 and OAD 152; Lane 6, Multiplex PCR of KRAS exon 2 and EGFR exons 18-21 with cocktail of primers used in Lanes 1–5. Concatenated KRAS and EGFR (CRE) product of ~915 bp amplified with OAD 176 and OAD 152 using multiplex PCR product as template derived from NCI-H1975 genomic DNA (shown in Panel B , Lane 2); derived from fresh frozen primary tumor genomic DNA (shown in Panel C , Lane 2); using tumor genomic DNA extracted from FFPE block (shown in Panel D, Lane 2).

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Derivative Assay, Formalin-fixed Paraffin-Embedded, Blocking Assay

    20) Product Images from "Tumor Content Chart-Assisted HER2/CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens"

    Article Title: Tumor Content Chart-Assisted HER2/CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154430

    HER2 -DISH in cell blocks and TC chart in the stepwise mixture system. (A–C) Micrographs of HER2 -DISH of LCL, H522, and SK-BR-3 FFPE cell blocks. HER2 signals are shown as black dots or clusters, and CEP17 signals are shown as red dots. HER2 and CEP17 copy numbers measured by HER2 -DISH and digital PCR are shown in Table 2 . (A) The LCL was genomically stable with two pairs of signals corresponding to HER2 and CEP17, mimicking non-cancerous stromal or inflammatory cells. (B) In H522 cells, both HER2 and CEP17 signals appeared as dots and individual signals were discernible. (C) In SK-BR-3 cells, HER2 signals formed clusters and exact measurements were difficult. CEP17 signals varied between 3 and 7, with an average score of 83/20 = 4.15. (D, E) Genomic DNA of H522 and SK-BR-3 cells was mixed with the LCL genome in a stepwise manner and analyzed by digital PCR. The horizontal axis represents the ratio of H522 or SK-BR-3 to LCL, corresponding to TCR [ x ] in clinical cases; the vertical axis represents the ratio of HER2 to CEP17 in digital PCR [ r ]. [ A ] and [ B ] were determined by HER2 -DISH ( Table 1 ). (D) In H522, [ A = 2.05] and [ B = 4.25], yielding [ r = (4.25 x + 2(1 − x ))/(2.05 x + 2(1 − x ))] (solid gray line). Plotted data were approximated by the theoretical line. (E) In SK-BR-3 cells, [ A = 4.15], but HER2 copy number [ B ] was difficult to determine due to cluster formation. HER2 copy number [ B′ ] was predicted [ B′ = 5.69 × 4.15 = 23.61] based on digital PCR data [ r ] and CEP17 copy number [ A ]. The equation [ r = (23.61 x + 2(1 − x ))/(4.15 x + 2(1 − x ))] is represented by the solid gray line. Plotted data were approximated by the theoretical line.
    Figure Legend Snippet: HER2 -DISH in cell blocks and TC chart in the stepwise mixture system. (A–C) Micrographs of HER2 -DISH of LCL, H522, and SK-BR-3 FFPE cell blocks. HER2 signals are shown as black dots or clusters, and CEP17 signals are shown as red dots. HER2 and CEP17 copy numbers measured by HER2 -DISH and digital PCR are shown in Table 2 . (A) The LCL was genomically stable with two pairs of signals corresponding to HER2 and CEP17, mimicking non-cancerous stromal or inflammatory cells. (B) In H522 cells, both HER2 and CEP17 signals appeared as dots and individual signals were discernible. (C) In SK-BR-3 cells, HER2 signals formed clusters and exact measurements were difficult. CEP17 signals varied between 3 and 7, with an average score of 83/20 = 4.15. (D, E) Genomic DNA of H522 and SK-BR-3 cells was mixed with the LCL genome in a stepwise manner and analyzed by digital PCR. The horizontal axis represents the ratio of H522 or SK-BR-3 to LCL, corresponding to TCR [ x ] in clinical cases; the vertical axis represents the ratio of HER2 to CEP17 in digital PCR [ r ]. [ A ] and [ B ] were determined by HER2 -DISH ( Table 1 ). (D) In H522, [ A = 2.05] and [ B = 4.25], yielding [ r = (4.25 x + 2(1 − x ))/(2.05 x + 2(1 − x ))] (solid gray line). Plotted data were approximated by the theoretical line. (E) In SK-BR-3 cells, [ A = 4.15], but HER2 copy number [ B ] was difficult to determine due to cluster formation. HER2 copy number [ B′ ] was predicted [ B′ = 5.69 × 4.15 = 23.61] based on digital PCR data [ r ] and CEP17 copy number [ A ]. The equation [ r = (23.61 x + 2(1 − x ))/(4.15 x + 2(1 − x ))] is represented by the solid gray line. Plotted data were approximated by the theoretical line.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Digital PCR

    21) Product Images from "Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus"

    Article Title: Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142379

    MLPA probe performance in gDNA from matched PBMC and FFPE-derived material. To evaluate the effects of FFPE treatment on MLPA performance, the variability of FcγR-specific probes was compared in (A) matched DNA from PBMCs and FFPE material. (B) The quality of FFPE material and its effect on FCGR probe MLPA performance was assessed by stratification of FFPE samples according to BIOMED-2 PCR fragment results. (C) The effects of the FFPE treatment process on the MLPA control probes were also assessed. Probes are represented in locus order with exons in brackets. A normalised peak height ratio of 1 represents a diploid copy number. Error bars represent mean +/- SD.
    Figure Legend Snippet: MLPA probe performance in gDNA from matched PBMC and FFPE-derived material. To evaluate the effects of FFPE treatment on MLPA performance, the variability of FcγR-specific probes was compared in (A) matched DNA from PBMCs and FFPE material. (B) The quality of FFPE material and its effect on FCGR probe MLPA performance was assessed by stratification of FFPE samples according to BIOMED-2 PCR fragment results. (C) The effects of the FFPE treatment process on the MLPA control probes were also assessed. Probes are represented in locus order with exons in brackets. A normalised peak height ratio of 1 represents a diploid copy number. Error bars represent mean +/- SD.

    Techniques Used: Multiplex Ligation-dependent Probe Amplification, Formalin-fixed Paraffin-Embedded, Derivative Assay, Polymerase Chain Reaction

    TaqMan allelic discrimination of FcγR SNPs in matched PBMC- and FFPE-derived material. TaqMan allelic discrimination assays were performed and compared in matched DNA from PBMC- (A, C, E) and FFPE- (B, D, F) derived material in n = 9 cases. Assays were performed in triplicate for SNPs (A-B) FCGR2A -131H/R rs1801274, (C-D) FCGR3A -158F/V rs396991 and (E-F) FCGR2B -232I/T rs1050501. All samples were performed in triplicate with non-template controls (NTCs). Axes represent relative fluorescence units (RFU). An outlier sample is defined as one where it was not possible to accurately genotype using Rotor-Gene Q software.
    Figure Legend Snippet: TaqMan allelic discrimination of FcγR SNPs in matched PBMC- and FFPE-derived material. TaqMan allelic discrimination assays were performed and compared in matched DNA from PBMC- (A, C, E) and FFPE- (B, D, F) derived material in n = 9 cases. Assays were performed in triplicate for SNPs (A-B) FCGR2A -131H/R rs1801274, (C-D) FCGR3A -158F/V rs396991 and (E-F) FCGR2B -232I/T rs1050501. All samples were performed in triplicate with non-template controls (NTCs). Axes represent relative fluorescence units (RFU). An outlier sample is defined as one where it was not possible to accurately genotype using Rotor-Gene Q software.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Fluorescence, Software

    22) Product Images from "Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA"

    Article Title: Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2015.281

    Patient 7. DNAs extracted from urine provide improved quality genomic data and clearer characterisation of the tumour profile than DNA from FFPE tumour material, despite repeat slides being cut and extracted. Two TARGET aberrations (CCND1 amplification (may predict sensitivity to CDK4/6 inhibitors) and CCNE1 amplification (may predict sensitivity to CDK2 inhibitors)) were observed in both cfDNA and urinary cellular DNA however none of these aberrations were independently called in two separate DNAs from FFPE tumour material.
    Figure Legend Snippet: Patient 7. DNAs extracted from urine provide improved quality genomic data and clearer characterisation of the tumour profile than DNA from FFPE tumour material, despite repeat slides being cut and extracted. Two TARGET aberrations (CCND1 amplification (may predict sensitivity to CDK4/6 inhibitors) and CCNE1 amplification (may predict sensitivity to CDK2 inhibitors)) were observed in both cfDNA and urinary cellular DNA however none of these aberrations were independently called in two separate DNAs from FFPE tumour material.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Amplification

    23) Product Images from "Differentially Expressed MicroRNAs in Postpartum Breast Cancer in Hispanic Women"

    Article Title: Differentially Expressed MicroRNAs in Postpartum Breast Cancer in Hispanic Women

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124340

    DNA Methylation of selected miRNAs. Box plots illustrating the DNA methylation of the miRNA genes investigated. The names of the miRNA MassARRAY amplicons analyzed by Sequenom are displayed at the top of each plot. The p-value (Wilcoxon test) for each amplicon analyzed is displayed on the second line at the top of the plot. The x-axis is divided into the two interval groups; early, representing Ella FFPE samples ≤ 5.2 years postpartum, and late, representing Ella FFPE samples ≥ 5.3 years postpartum. The y-axis represents the mean DNA methylation (%) of the amplicon.
    Figure Legend Snippet: DNA Methylation of selected miRNAs. Box plots illustrating the DNA methylation of the miRNA genes investigated. The names of the miRNA MassARRAY amplicons analyzed by Sequenom are displayed at the top of each plot. The p-value (Wilcoxon test) for each amplicon analyzed is displayed on the second line at the top of the plot. The x-axis is divided into the two interval groups; early, representing Ella FFPE samples ≤ 5.2 years postpartum, and late, representing Ella FFPE samples ≥ 5.3 years postpartum. The y-axis represents the mean DNA methylation (%) of the amplicon.

    Techniques Used: DNA Methylation Assay, Amplification, Formalin-fixed Paraffin-Embedded

    24) Product Images from "A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue"

    Article Title: A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    Journal: Analytical Biochemistry

    doi: 10.1016/j.ab.2012.03.012

    Schematic diagram of DNA extraction procedures from FFPE tissue section or core.
    Figure Legend Snippet: Schematic diagram of DNA extraction procedures from FFPE tissue section or core.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Analysis of the quality of the DNA extracted from archival human colon FFPE tissues by the Nanodrop spectrophotometer. The DNA quality was accessed by distribution of the ratio values (A 260 /A 280 and A 260 /A 230 ).
    Figure Legend Snippet: Analysis of the quality of the DNA extracted from archival human colon FFPE tissues by the Nanodrop spectrophotometer. The DNA quality was accessed by distribution of the ratio values (A 260 /A 280 and A 260 /A 230 ).

    Techniques Used: Formalin-fixed Paraffin-Embedded, Spectrophotometry

    Comparison of DNA extraction yield and quality between a new rapid method and the QIAamp DNA FFPE tissue kit. We performed DNA extraction from archival human liver cores or sections. Representative data were presented as a bar graph ( A ) and a gel image
    Figure Legend Snippet: Comparison of DNA extraction yield and quality between a new rapid method and the QIAamp DNA FFPE tissue kit. We performed DNA extraction from archival human liver cores or sections. Representative data were presented as a bar graph ( A ) and a gel image

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    MSP analysis of sFRP1 and TFPI2 genes in colon samples using the rapid and classic DNA extraction. Methylation pattern of (A) sFRP1 and (B) TFPI2 gene promoter region in 18 FFPE colon samples using rapid DNA extraction method. Colon FFPE tumor samples
    Figure Legend Snippet: MSP analysis of sFRP1 and TFPI2 genes in colon samples using the rapid and classic DNA extraction. Methylation pattern of (A) sFRP1 and (B) TFPI2 gene promoter region in 18 FFPE colon samples using rapid DNA extraction method. Colon FFPE tumor samples

    Techniques Used: DNA Extraction, Methylation, Formalin-fixed Paraffin-Embedded

    25) Product Images from "A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue"

    Article Title: A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    Journal: Analytical Biochemistry

    doi: 10.1016/j.ab.2012.03.012

    Schematic diagram of DNA extraction procedures from FFPE tissue section or core.
    Figure Legend Snippet: Schematic diagram of DNA extraction procedures from FFPE tissue section or core.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Analysis of the quality of the DNA extracted from archival human colon FFPE tissues by the Nanodrop spectrophotometer. The DNA quality was accessed by distribution of the ratio values (A 260 /A 280 and A 260 /A 230 ).
    Figure Legend Snippet: Analysis of the quality of the DNA extracted from archival human colon FFPE tissues by the Nanodrop spectrophotometer. The DNA quality was accessed by distribution of the ratio values (A 260 /A 280 and A 260 /A 230 ).

    Techniques Used: Formalin-fixed Paraffin-Embedded, Spectrophotometry

    Comparison of DNA extraction yield and quality between a new rapid method and the QIAamp DNA FFPE tissue kit. We performed DNA extraction from archival human liver cores or sections. Representative data were presented as a bar graph ( A ) and a gel image
    Figure Legend Snippet: Comparison of DNA extraction yield and quality between a new rapid method and the QIAamp DNA FFPE tissue kit. We performed DNA extraction from archival human liver cores or sections. Representative data were presented as a bar graph ( A ) and a gel image

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    MSP analysis of sFRP1 and TFPI2 genes in colon samples using the rapid and classic DNA extraction. Methylation pattern of (A) sFRP1 and (B) TFPI2 gene promoter region in 18 FFPE colon samples using rapid DNA extraction method. Colon FFPE tumor samples
    Figure Legend Snippet: MSP analysis of sFRP1 and TFPI2 genes in colon samples using the rapid and classic DNA extraction. Methylation pattern of (A) sFRP1 and (B) TFPI2 gene promoter region in 18 FFPE colon samples using rapid DNA extraction method. Colon FFPE tumor samples

    Techniques Used: DNA Extraction, Methylation, Formalin-fixed Paraffin-Embedded

    26) Product Images from "Establishment and characterization of an oral tongue squamous cell carcinoma cell line from a never-smoking patient"

    Article Title: Establishment and characterization of an oral tongue squamous cell carcinoma cell line from a never-smoking patient

    Journal: Oral oncology

    doi: 10.1016/j.oraloncology.2017.03.020

    TP53-targeted sequencing of UCSF-OT-1109 (A, forward read; B, reverse read) and the primary tumor (D, forward read; E, reverse read) (C) Western blot analysis with p53 (short and long exposure) and β-Actin antibodies: Lanes 1–5, UCSF-OT-1109 clone; Lanes 6–8, OTSCC lines, Lane 9, cervical cancer cell line HeLa; Lane 10, colon cancer cell line SW480. In D and E, genomic DNA was isolated from microdissected FFPE tissue sections.
    Figure Legend Snippet: TP53-targeted sequencing of UCSF-OT-1109 (A, forward read; B, reverse read) and the primary tumor (D, forward read; E, reverse read) (C) Western blot analysis with p53 (short and long exposure) and β-Actin antibodies: Lanes 1–5, UCSF-OT-1109 clone; Lanes 6–8, OTSCC lines, Lane 9, cervical cancer cell line HeLa; Lane 10, colon cancer cell line SW480. In D and E, genomic DNA was isolated from microdissected FFPE tissue sections.

    Techniques Used: Sequencing, Western Blot, Isolation, Formalin-fixed Paraffin-Embedded

    27) Product Images from "Personalized genomic analysis based on circulating tumor cells of extra-skeletal Ewing sarcoma of the uterus: A case report of a 16-year-old Korean female"

    Article Title: Personalized genomic analysis based on circulating tumor cells of extra-skeletal Ewing sarcoma of the uterus: A case report of a 16-year-old Korean female

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6323

    Molecular pathology findings of Ewing sarcoma tissue. (A) EWS-FLI1 fusion transcript was detected in the patient's FFPE DNA. (B) Translocation breakpoint of the EWS-FLI1 fusion transcript was observed in the patient's FFPE sample DNA. FFPE, formalin-fixed paraffin-embedded.
    Figure Legend Snippet: Molecular pathology findings of Ewing sarcoma tissue. (A) EWS-FLI1 fusion transcript was detected in the patient's FFPE DNA. (B) Translocation breakpoint of the EWS-FLI1 fusion transcript was observed in the patient's FFPE sample DNA. FFPE, formalin-fixed paraffin-embedded.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Translocation Assay

    28) Product Images from "Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice"

    Article Title: Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196434

    Read quality in the baseline group does not change by DNA extraction type. Read quality as measured by percent reads mapped. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Read quality in the baseline group does not change by DNA extraction type. Read quality as measured by percent reads mapped. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Deamination events increase with sample age when not treated with UNG. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Deamination events increase with sample age when not treated with UNG. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    Total reads mapped in the baseline group does not change by DNA extraction type. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Total reads mapped in the baseline group does not change by DNA extraction type. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Sequencing read quality decreases over fixation time. Read quality as a measure of percent reads mapped (y-axis) decreases with a longer tissue fixation time (x-axis). Blue dots represent DNA without UNG treatment (extracted using QiaAmp FFPE kit; QA), orange crossed represent matched DNA treated with UNG (extraction using the GeneRead kit; GR UNG). Linear regression line depicted by the central broken line and encompassed by dotted lines representing the 95% confidence interval (CI region colored). Frozen samples used for ground truth are represented by green dots. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Sequencing read quality decreases over fixation time. Read quality as a measure of percent reads mapped (y-axis) decreases with a longer tissue fixation time (x-axis). Blue dots represent DNA without UNG treatment (extracted using QiaAmp FFPE kit; QA), orange crossed represent matched DNA treated with UNG (extraction using the GeneRead kit; GR UNG). Linear regression line depicted by the central broken line and encompassed by dotted lines representing the 95% confidence interval (CI region colored). Frozen samples used for ground truth are represented by green dots. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

    Read quality decreases with sample age in the age group. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Read quality decreases with sample age in the age group. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    29) Product Images from "Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent"

    Article Title: Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.14741.2

    DNA methylation across the body of the HTLV-1 provirus. ( a ) Upper panel: count of CpG dinucleotides in a window of 350 bp in the HTLV-1 reference genome (L36905). Lower panel: schematic diagram of HTLV-1 provirus indicating the two LTRs and the 9 loci examined by MeDIP. ( b ) DNA methylation on the HTLV-1 provirus in the Tax + and Tax – populations from two HTLV-1-infected T cell clones (Clones TBX4B and 11.65). ( c ) DNA methylation on the HTLV-1 provirus in the CADM1 + Tax + and CADM1 + Tax – populations in PBMCs from two unrelated individuals (Patients TDZ and TED). The asterisk (*) indicates that the PCR failed to amplify.
    Figure Legend Snippet: DNA methylation across the body of the HTLV-1 provirus. ( a ) Upper panel: count of CpG dinucleotides in a window of 350 bp in the HTLV-1 reference genome (L36905). Lower panel: schematic diagram of HTLV-1 provirus indicating the two LTRs and the 9 loci examined by MeDIP. ( b ) DNA methylation on the HTLV-1 provirus in the Tax + and Tax – populations from two HTLV-1-infected T cell clones (Clones TBX4B and 11.65). ( c ) DNA methylation on the HTLV-1 provirus in the CADM1 + Tax + and CADM1 + Tax – populations in PBMCs from two unrelated individuals (Patients TDZ and TED). The asterisk (*) indicates that the PCR failed to amplify.

    Techniques Used: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Infection, Clone Assay, Polymerase Chain Reaction

    DNA methylation in the HTLV-1 LTR of patient-derived PBMCs. ( a ) The HTLV-1 LTR sequence (Accession no. L36905) with CpG dinucleotides highlighted in bold. The three TREs are coloured in red; the TATA box is indicated in the rectangle. ( b ) Schematic diagram of HTLV-1 provirus and the regions amplified with indicated sets of primers for bisulfite-sequencing. Sequencing results for each region are shown in the corresponding panels ( c – f ). The three TREs are indicated by red bars. ( c – f ) Schematic representation of DNA methylation for each clone sequenced. Open circles indicate unmethylated cytosine; closed circles methylated cytosine. The numbers indicate the corresponding CpG sites in panel ( a ).
    Figure Legend Snippet: DNA methylation in the HTLV-1 LTR of patient-derived PBMCs. ( a ) The HTLV-1 LTR sequence (Accession no. L36905) with CpG dinucleotides highlighted in bold. The three TREs are coloured in red; the TATA box is indicated in the rectangle. ( b ) Schematic diagram of HTLV-1 provirus and the regions amplified with indicated sets of primers for bisulfite-sequencing. Sequencing results for each region are shown in the corresponding panels ( c – f ). The three TREs are indicated by red bars. ( c – f ) Schematic representation of DNA methylation for each clone sequenced. Open circles indicate unmethylated cytosine; closed circles methylated cytosine. The numbers indicate the corresponding CpG sites in panel ( a ).

    Techniques Used: DNA Methylation Assay, Derivative Assay, Sequencing, Amplification, Methylation Sequencing, Methylation

    Overview of the cell preparations. ( a ) Preparation of Tax + and Tax – populations from PBMCs obtained from HTLV-1-infected patients. PBMCs were stained for CD4, CADM1 and Tax after overnight culture. Tax + and Tax – fractions were collected from the CADM1 + population. ( b ) Preparation of the Tax + and Tax – populations from HTLV-1-infected T cell clones. HTLV-1-infected T cell clones were stained for intracellular Tax and sorted according to Tax expression.
    Figure Legend Snippet: Overview of the cell preparations. ( a ) Preparation of Tax + and Tax – populations from PBMCs obtained from HTLV-1-infected patients. PBMCs were stained for CD4, CADM1 and Tax after overnight culture. Tax + and Tax – fractions were collected from the CADM1 + population. ( b ) Preparation of the Tax + and Tax – populations from HTLV-1-infected T cell clones. HTLV-1-infected T cell clones were stained for intracellular Tax and sorted according to Tax expression.

    Techniques Used: Infection, Staining, Clone Assay, Expressing

    HTLV-1 transcription in two distinct models. a ) Schematic diagram of HTLV-1 provirus inserted in the host genome. The HTLV-1 provirus has two identical LTRs, one at each end of the provirus. As well as genes encoding the canonical retroviral structural components Gag, Pol and Env, the provirus contains a group of regulatory genes in the pX region on the plus-strand. The plus-strand transcripts, represented by tax , are coloured in red, and the minus-strand transcript HBZ in yellow. ( b ) In PBMCs freshly isolated from HTLV-1 carriers, HTLV-1 reactivates and expresses the plus-strand transcripts within a few hours of culture; but these transcripts remain transcriptionally silent for most of the time in vivo . ( c ) In HTLV-1-infected T cell clones cultured in vitro , the promoter activity for plus-strand transcripts shuttles between the on and off state. The plus-strand transcripts are only produced when the promoter activity is on, yielding only a limited fraction of cells that are positive for the plus-strand transcripts at a given time.
    Figure Legend Snippet: HTLV-1 transcription in two distinct models. a ) Schematic diagram of HTLV-1 provirus inserted in the host genome. The HTLV-1 provirus has two identical LTRs, one at each end of the provirus. As well as genes encoding the canonical retroviral structural components Gag, Pol and Env, the provirus contains a group of regulatory genes in the pX region on the plus-strand. The plus-strand transcripts, represented by tax , are coloured in red, and the minus-strand transcript HBZ in yellow. ( b ) In PBMCs freshly isolated from HTLV-1 carriers, HTLV-1 reactivates and expresses the plus-strand transcripts within a few hours of culture; but these transcripts remain transcriptionally silent for most of the time in vivo . ( c ) In HTLV-1-infected T cell clones cultured in vitro , the promoter activity for plus-strand transcripts shuttles between the on and off state. The plus-strand transcripts are only produced when the promoter activity is on, yielding only a limited fraction of cells that are positive for the plus-strand transcripts at a given time.

    Techniques Used: Isolation, In Vivo, Infection, Clone Assay, Cell Culture, In Vitro, Activity Assay, Produced

    Kinetics of the plus- and minus-strand transcription in HTLV-1-infected T cell clones. ( a ) Representative images of HTLV-1 transcripts by single-molecule RNA-FISH (maximum-projection of Z-stacks). Red spots indicate the plus-strand transcripts, and yellow spots the minus-strand transcripts. Blue indicates the DAPI-stained nucleus. Plus- and minus-signs in brackets indicate respectively the presence or absence of the mRNA. Scale bar (white) = 5 µm. ( b ) Spot counts of the plus-strand (upper row) and the minus-strand transcripts (lower row) respectively in the unaltered and ΔCTCF-binding subclones. The insets in the upper row capture low-frequency events on a magnified y-axis. The bar in the first bin in the insets is greyed out because it is out of scale.
    Figure Legend Snippet: Kinetics of the plus- and minus-strand transcription in HTLV-1-infected T cell clones. ( a ) Representative images of HTLV-1 transcripts by single-molecule RNA-FISH (maximum-projection of Z-stacks). Red spots indicate the plus-strand transcripts, and yellow spots the minus-strand transcripts. Blue indicates the DAPI-stained nucleus. Plus- and minus-signs in brackets indicate respectively the presence or absence of the mRNA. Scale bar (white) = 5 µm. ( b ) Spot counts of the plus-strand (upper row) and the minus-strand transcripts (lower row) respectively in the unaltered and ΔCTCF-binding subclones. The insets in the upper row capture low-frequency events on a magnified y-axis. The bar in the first bin in the insets is greyed out because it is out of scale.

    Techniques Used: Infection, Clone Assay, Fluorescence In Situ Hybridization, Staining, Binding Assay

    Histone modifications and CTCF-binding in the HTLV-1 provirus. Chromatin immunoprecipitation (ChIP) was used to identify ( a ) histone modifications and CTCF-binding in the Tax + and Tax – populations from an HTLV-1-infected T cell clone (TBX4B); and ( b ) histone modifications in the CADM1 + Tax + and CADM1 + Tax – populations from PBMCs obtained from HTLV-1-infected patients (Patients TW and TCD). The horizontal axis indicates the nucleotide position in the full-length HTLV-1 provirus (J02029), and the vertical axis the read depth (arbitrary units). The reads that aligned within either one of the LTRs are greyed out. The black bars on the horizontal axis indicates the LTRs.
    Figure Legend Snippet: Histone modifications and CTCF-binding in the HTLV-1 provirus. Chromatin immunoprecipitation (ChIP) was used to identify ( a ) histone modifications and CTCF-binding in the Tax + and Tax – populations from an HTLV-1-infected T cell clone (TBX4B); and ( b ) histone modifications in the CADM1 + Tax + and CADM1 + Tax – populations from PBMCs obtained from HTLV-1-infected patients (Patients TW and TCD). The horizontal axis indicates the nucleotide position in the full-length HTLV-1 provirus (J02029), and the vertical axis the read depth (arbitrary units). The reads that aligned within either one of the LTRs are greyed out. The black bars on the horizontal axis indicates the LTRs.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Infection

    Epigenetic modifications in the HTLV-1 provirus lacking CTCF binding. ( a ) Histone modifications in the Tax + and Tax – populations of the altered HTLV-1-infected T cell clone (Subclone #78 of TBX4B). ( b ) DNA methylation in the body of the provirus in TBX4B-78. Note the similarity to the profiles of epigenetic modifications in the wild-type TBX4B ( Figure 3 ).
    Figure Legend Snippet: Epigenetic modifications in the HTLV-1 provirus lacking CTCF binding. ( a ) Histone modifications in the Tax + and Tax – populations of the altered HTLV-1-infected T cell clone (Subclone #78 of TBX4B). ( b ) DNA methylation in the body of the provirus in TBX4B-78. Note the similarity to the profiles of epigenetic modifications in the wild-type TBX4B ( Figure 3 ).

    Techniques Used: Binding Assay, Infection, DNA Methylation Assay

    CTCF occupancy in the HTLV-1 provirus in patient-derived PBMCs. CTCF occupancy was examined by droplet digital PCR following ChIP for CTCF. The experiment was carried out on PBMCs after overnight incubation in vitro . Replicate 1 is obtained from pooled samples of 4 patients (TCR, TEJ, TED and TW) and Replicate 2 from 3 patients (TED, TCR and TEJ).
    Figure Legend Snippet: CTCF occupancy in the HTLV-1 provirus in patient-derived PBMCs. CTCF occupancy was examined by droplet digital PCR following ChIP for CTCF. The experiment was carried out on PBMCs after overnight incubation in vitro . Replicate 1 is obtained from pooled samples of 4 patients (TCR, TEJ, TED and TW) and Replicate 2 from 3 patients (TED, TCR and TEJ).

    Techniques Used: Derivative Assay, Digital PCR, Chromatin Immunoprecipitation, Incubation, In Vitro

    Alteration of the CTCF-binding site in the provirus in HTLV-1-infected T cell clones. ( a ) The sequence of the CTCF-binding site in the HTLV-1 provirus. The upper panel is from a subclone with the sequence unchanged, and the lower panel from a subclone in which the sequence was altered by CRISPR/Cas9 modification. ( b ) Flow cytometric analysis of the mutated clone after staining for intracellular Tax protein.
    Figure Legend Snippet: Alteration of the CTCF-binding site in the provirus in HTLV-1-infected T cell clones. ( a ) The sequence of the CTCF-binding site in the HTLV-1 provirus. The upper panel is from a subclone with the sequence unchanged, and the lower panel from a subclone in which the sequence was altered by CRISPR/Cas9 modification. ( b ) Flow cytometric analysis of the mutated clone after staining for intracellular Tax protein.

    Techniques Used: Binding Assay, Infection, Clone Assay, Sequencing, CRISPR, Modification, Flow Cytometry, Staining

    30) Product Images from "Profiling Cancer Gene Mutations in Clinical Formalin-Fixed, Paraffin-Embedded Colorectal Tumor Specimens Using Targeted Next-Generation Sequencing"

    Article Title: Profiling Cancer Gene Mutations in Clinical Formalin-Fixed, Paraffin-Embedded Colorectal Tumor Specimens Using Targeted Next-Generation Sequencing

    Journal: The Oncologist

    doi: 10.1634/theoncologist.2013-0180

    Mutation detection using formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tumor specimens. Variants were detected by AmpliSeq (A) and SimpliSeq (B) in 44 FFPE genomic DNA samples. The red shade stands for variants with VF > 5%; blue shade
    Figure Legend Snippet: Mutation detection using formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tumor specimens. Variants were detected by AmpliSeq (A) and SimpliSeq (B) in 44 FFPE genomic DNA samples. The red shade stands for variants with VF > 5%; blue shade

    Techniques Used: Mutagenesis, Formalin-fixed Paraffin-Embedded

    31) Product Images from "Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study"

    Article Title: Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21256

    Optimization of circulating free DNA (cfDNA) extraction and quantification of cfDNA in the samples (A) Reproducibility of cfDNA extraction using the QIAamp Circulating Acid Kit (Qiagen, Cat No 55114, Valencia, CA, USA) on two independent cfDNA samples extracted from 1 mL (Ai) and 3 mL (Aii) of plasma from NSCLC patients. After extraction, cfDNA was quantified by Qubit dsDNA HS Assay Kit (Life Technologies, Q32854, Carlsbad, CA, USA) according to the manufacturer's instructions. (B) Correlation between the initial volume of plasma 1 mL versus 3 mL (Bi) or 3 mL versus 5 mL (Bii) and the quantity of cfDNA extracted (in ng/μL). (Ci) Fragment size visualization of cfDNA (in bp) from a concentrated (left) and a less concentrated (right) sample obtained using the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (Cii) , and average size distribution (10 bp increments) of cfDNA fragments in 77 plasma samples. (D) Correlation between cfDNA concentration measured using the Qubit method and the number of amplifiable copies in the corresponding plasma samples determined using the Quantifiler Kit.
    Figure Legend Snippet: Optimization of circulating free DNA (cfDNA) extraction and quantification of cfDNA in the samples (A) Reproducibility of cfDNA extraction using the QIAamp Circulating Acid Kit (Qiagen, Cat No 55114, Valencia, CA, USA) on two independent cfDNA samples extracted from 1 mL (Ai) and 3 mL (Aii) of plasma from NSCLC patients. After extraction, cfDNA was quantified by Qubit dsDNA HS Assay Kit (Life Technologies, Q32854, Carlsbad, CA, USA) according to the manufacturer's instructions. (B) Correlation between the initial volume of plasma 1 mL versus 3 mL (Bi) or 3 mL versus 5 mL (Bii) and the quantity of cfDNA extracted (in ng/μL). (Ci) Fragment size visualization of cfDNA (in bp) from a concentrated (left) and a less concentrated (right) sample obtained using the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (Cii) , and average size distribution (10 bp increments) of cfDNA fragments in 77 plasma samples. (D) Correlation between cfDNA concentration measured using the Qubit method and the number of amplifiable copies in the corresponding plasma samples determined using the Quantifiler Kit.

    Techniques Used: Concentration Assay

    32) Product Images from "Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples"

    Article Title: Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195471

    DNA Integrity Number (DIN) values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.
    Figure Legend Snippet: DNA Integrity Number (DIN) values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    DNA fragment length values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.
    Figure Legend Snippet: DNA fragment length values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    33) Product Images from "Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples"

    Article Title: Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195471

    DNA Integrity Number (DIN) values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.
    Figure Legend Snippet: DNA Integrity Number (DIN) values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    DNA fragment length values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.
    Figure Legend Snippet: DNA fragment length values for FF and FFPE samples. A: FF and FFPE samples. B: FFPE samples grouped by extraction method.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    34) Product Images from "Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples"

    Article Title: Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197456

    Variants identified in the samples extracted with different DNA extraction methods. (A-D) Venn diagram showing the distribution of variants between DNA extraction methods in four different FFPE samples, SARC1-4. Variants were detected by MuTect and Strelka, using an artificial control as normal. The data was filtered on exonic variants with allele frequency > 5% and coverage > 100x, being present at
    Figure Legend Snippet: Variants identified in the samples extracted with different DNA extraction methods. (A-D) Venn diagram showing the distribution of variants between DNA extraction methods in four different FFPE samples, SARC1-4. Variants were detected by MuTect and Strelka, using an artificial control as normal. The data was filtered on exonic variants with allele frequency > 5% and coverage > 100x, being present at

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Yield and amplifiability of extracted DNA and RNA. (A) Average total amount of DNA. (B) Average total amount of RNA. (C) Amplifiable DNA quantified with the FFPE QC kit from Illumina. (D) Amplifiable RNA quantified with the PreSeq QC assay from ArcherDx. The average total amount and average delta Ct values for the different samples and extraction methods are shown. The standard deviation is shown as vertical bars. Methods with significant differences in yield are marked as connected with horizontal bars (p
    Figure Legend Snippet: Yield and amplifiability of extracted DNA and RNA. (A) Average total amount of DNA. (B) Average total amount of RNA. (C) Amplifiable DNA quantified with the FFPE QC kit from Illumina. (D) Amplifiable RNA quantified with the PreSeq QC assay from ArcherDx. The average total amount and average delta Ct values for the different samples and extraction methods are shown. The standard deviation is shown as vertical bars. Methods with significant differences in yield are marked as connected with horizontal bars (p

    Techniques Used: Formalin-fixed Paraffin-Embedded, Standard Deviation

    35) Product Images from "Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA"

    Article Title: Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA

    Journal: American Journal of Clinical Pathology

    doi: 10.1093/ajcp/aqw205

    The influence of five different DNA extraction methods on the multiplex ligation-dependent probe amplification (MLPA) probe’s copy number ratios. MLPA was performed on DNA extracted from eight various formalin-fixed, paraffin-embedded (FFPE) tissues with the P027 probe mix (50 probes). The y-axis represents the percentage of probes showing copy number ratios outside the 0.8 to 1.2 normal copy number range. The x-axis shows five different DNA extraction methods. Each bar represents a separate FFPE tissue-type block. For samples where DNA was extracted with the one-tube FFPE extraction method, results from crude (nonpurified) DNA lysate are presented for all tissues, except the lung. The crude lysate of the lung FFPE tissue was also purified. SEM (as indicated by error bars) was calculated by dividing the standard deviation of the mean of the number of probes with copy number ratios outside the normal range by the square root of the number of samples (triplicate) ( Supplementary Table S6 ). colonSymb, tissue from Symbiant Pathology Expert Centre, Alkmaar; colonUMC, tissue from Department of Pathology, University Medical Centre Utrecht, Utrecht.
    Figure Legend Snippet: The influence of five different DNA extraction methods on the multiplex ligation-dependent probe amplification (MLPA) probe’s copy number ratios. MLPA was performed on DNA extracted from eight various formalin-fixed, paraffin-embedded (FFPE) tissues with the P027 probe mix (50 probes). The y-axis represents the percentage of probes showing copy number ratios outside the 0.8 to 1.2 normal copy number range. The x-axis shows five different DNA extraction methods. Each bar represents a separate FFPE tissue-type block. For samples where DNA was extracted with the one-tube FFPE extraction method, results from crude (nonpurified) DNA lysate are presented for all tissues, except the lung. The crude lysate of the lung FFPE tissue was also purified. SEM (as indicated by error bars) was calculated by dividing the standard deviation of the mean of the number of probes with copy number ratios outside the normal range by the square root of the number of samples (triplicate) ( Supplementary Table S6 ). colonSymb, tissue from Symbiant Pathology Expert Centre, Alkmaar; colonUMC, tissue from Department of Pathology, University Medical Centre Utrecht, Utrecht.

    Techniques Used: DNA Extraction, Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification, Formalin-fixed Paraffin-Embedded, Blocking Assay, Purification, Standard Deviation

    (cont) C , MLPA electropherogram of FFPE lung tissue crude lysate column purified with the DNA Clean Concentrator-5 kit. RFU, residual fluorescence unit.
    Figure Legend Snippet: (cont) C , MLPA electropherogram of FFPE lung tissue crude lysate column purified with the DNA Clean Concentrator-5 kit. RFU, residual fluorescence unit.

    Techniques Used: Multiplex Ligation-dependent Probe Amplification, Formalin-fixed Paraffin-Embedded, Purification, Fluorescence

    36) Product Images from "The frequency of promoter DNA hypermethylation is decreased in colorectal neoplasms of familial adenomatous polyposis"

    Article Title: The frequency of promoter DNA hypermethylation is decreased in colorectal neoplasms of familial adenomatous polyposis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25987

    Selection of appropriate probes for analysis of frozen and FFPE samples ( A ) Preparation of frozen and FFPE samples. Three colon tumors (#1–3) were cut into two pieces; one was fixed with formalin and embedded in paraffin ( FFPE ), and the other was frozen with liquid nitrogen and stored at –80° C ( frozen ). Both tissues underwent DNA extraction and Infinium assays. ( B ) Plot of β-values. Probes showing the differences in β-values between frozen and FFPE samples of less than 0.1, i.e., between y = x + 0.1 and y = x−0.1, were extracted ( red ). ( C ) In total, 161,828 overlapped probes among three analyses (tumors #1, #2, and #3) were extracted and used for subsequent DNA methylation analysis.
    Figure Legend Snippet: Selection of appropriate probes for analysis of frozen and FFPE samples ( A ) Preparation of frozen and FFPE samples. Three colon tumors (#1–3) were cut into two pieces; one was fixed with formalin and embedded in paraffin ( FFPE ), and the other was frozen with liquid nitrogen and stored at –80° C ( frozen ). Both tissues underwent DNA extraction and Infinium assays. ( B ) Plot of β-values. Probes showing the differences in β-values between frozen and FFPE samples of less than 0.1, i.e., between y = x + 0.1 and y = x−0.1, were extracted ( red ). ( C ) In total, 161,828 overlapped probes among three analyses (tumors #1, #2, and #3) were extracted and used for subsequent DNA methylation analysis.

    Techniques Used: Selection, Formalin-fixed Paraffin-Embedded, DNA Extraction, DNA Methylation Assay

    37) Product Images from "Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples"

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1142

    SSAR mapping and diagrammatic depiction of the proposed mechanism. The SSAR example shown is a screen shot of an actual IGV image. At the top (I) we depict a ds-DNA region of intact gDNA. In the process of FFPE preparation, storage and extraction (II), gDNA is fragmented and denatured. In the absence of S1 nuclease (III left), ss-DNA fragments from non-contiguous regions of the genome anneal via short complementary repetitive sequences (red asterisks). In contrast, ss-DNA fragments and overhangs are removed upon treatment with S1 nuclease (III right). During the end-repair step of library construction, T4 DNA polymerase removes overhangs (IV) and fills ends (V), resulting in the formation of double-stranded chimeric fragments (‘A’ in V). One class of such chimeric fragments yield SSARs (‘A’ in VI). R1 = read; R2 = read 2. For SSARs, part of Read 2 aligns in the expected paired-end orientation while the distal end of Read 2 does not match the reference at that position and instead aligns to a nearby region of the reference genome in the opposite orientation (denoted as R2′).
    Figure Legend Snippet: SSAR mapping and diagrammatic depiction of the proposed mechanism. The SSAR example shown is a screen shot of an actual IGV image. At the top (I) we depict a ds-DNA region of intact gDNA. In the process of FFPE preparation, storage and extraction (II), gDNA is fragmented and denatured. In the absence of S1 nuclease (III left), ss-DNA fragments from non-contiguous regions of the genome anneal via short complementary repetitive sequences (red asterisks). In contrast, ss-DNA fragments and overhangs are removed upon treatment with S1 nuclease (III right). During the end-repair step of library construction, T4 DNA polymerase removes overhangs (IV) and fills ends (V), resulting in the formation of double-stranded chimeric fragments (‘A’ in V). One class of such chimeric fragments yield SSARs (‘A’ in VI). R1 = read; R2 = read 2. For SSARs, part of Read 2 aligns in the expected paired-end orientation while the distal end of Read 2 does not match the reference at that position and instead aligns to a nearby region of the reference genome in the opposite orientation (denoted as R2′).

    Techniques Used: Formalin-fixed Paraffin-Embedded

    38) Product Images from "Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)"

    Article Title: Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150197

    Plasma input volume comparison. Three volumes of plasma (1 mL, 2 mL and 3 mL) were processed from each sample in a cohort of 15 NSCLC patients. DNA was extracted using the QIAamp CNA Kit and was measured by qPCR using the ABI TaqMan® RNase P Detection Reagent Kit. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p
    Figure Legend Snippet: Plasma input volume comparison. Three volumes of plasma (1 mL, 2 mL and 3 mL) were processed from each sample in a cohort of 15 NSCLC patients. DNA was extracted using the QIAamp CNA Kit and was measured by qPCR using the ABI TaqMan® RNase P Detection Reagent Kit. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p

    Techniques Used: Real-time Polymerase Chain Reaction

    DNA extraction kit comparison. Equal volumes of plasma (2 mL) from 10 NSCLC patients were processed using three different DNA extraction methods: QIAamp Circulating Nucleic Acid Kit (Qiagen CNA Kit); PME free-circulating DNA Extraction Kit (Analytik Jena) and the DSP Virus/Pathogen Midi Kit performed on QIAsymphony (QIAsymphony). DNA was measured by qPCR using the ABI TaqMan® RNase P Detection Reagent Kit. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p
    Figure Legend Snippet: DNA extraction kit comparison. Equal volumes of plasma (2 mL) from 10 NSCLC patients were processed using three different DNA extraction methods: QIAamp Circulating Nucleic Acid Kit (Qiagen CNA Kit); PME free-circulating DNA Extraction Kit (Analytik Jena) and the DSP Virus/Pathogen Midi Kit performed on QIAsymphony (QIAsymphony). DNA was measured by qPCR using the ABI TaqMan® RNase P Detection Reagent Kit. Results are displayed for each patient. Statistical analysis was performed using a paired Student’s t-test where; **p

    Techniques Used: DNA Extraction, Real-time Polymerase Chain Reaction

    39) Product Images from "Multi-Purpose Utility of Circulating Plasma DNA Testing in Patients with Advanced Cancers"

    Article Title: Multi-Purpose Utility of Circulating Plasma DNA Testing in Patients with Advanced Cancers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047020

    cpDNA concentrations for mutational detection by Sequenom OncoCarta panel (v1.0). 2A: Nonparametric ROC analyses were used to assess the limit of the Sequenom platform to detect OncoCarta panel mutations in cpDNA. Each dot on the graph corresponds to the sensitivity and specificity at one of the observed concentrations. Mutations were considered ‘available for detection’ if they were detected in the patient's FFPE tissue. Mutations were detected in FFPE samples from 37 patients. The concentration of cpDNA with the optimal ability to detect a mutation is 29.95 ng/ml (Likelihood ratio = 7.3043). The AUC calculated is 0.8075 (95% CI 0.6552–0.9598). Patients whose FFPE was unavailable or tested negative for mutations were excluded from the analysis. The specificity reference lines for quartiles of DNA concentrations are indicated in red dashed lines. 2B: Graph showing the types of mutations and cpDNA concentrations at which they were detected in different tumors. Mutations were detected in six oncogenes. Symbols represent different tumor types.
    Figure Legend Snippet: cpDNA concentrations for mutational detection by Sequenom OncoCarta panel (v1.0). 2A: Nonparametric ROC analyses were used to assess the limit of the Sequenom platform to detect OncoCarta panel mutations in cpDNA. Each dot on the graph corresponds to the sensitivity and specificity at one of the observed concentrations. Mutations were considered ‘available for detection’ if they were detected in the patient's FFPE tissue. Mutations were detected in FFPE samples from 37 patients. The concentration of cpDNA with the optimal ability to detect a mutation is 29.95 ng/ml (Likelihood ratio = 7.3043). The AUC calculated is 0.8075 (95% CI 0.6552–0.9598). Patients whose FFPE was unavailable or tested negative for mutations were excluded from the analysis. The specificity reference lines for quartiles of DNA concentrations are indicated in red dashed lines. 2B: Graph showing the types of mutations and cpDNA concentrations at which they were detected in different tumors. Mutations were detected in six oncogenes. Symbols represent different tumor types.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Concentration Assay, Mutagenesis

    40) Product Images from "Resectable lung lesions malignancy assessment and cancer detection by ultra-deep sequencing of targeted gene mutations in plasma cell-free DNA"

    Article Title: Resectable lung lesions malignancy assessment and cancer detection by ultra-deep sequencing of targeted gene mutations in plasma cell-free DNA

    Journal: Journal of Medical Genetics

    doi: 10.1136/jmedgenet-2018-105825

    Driver mutation distribution in patients with lung cancer patients. (A) Plasma ctDNA. (B) FFPE gDNA. ctDNA, circulating tumour DNA; gDNA, genomic DNA.
    Figure Legend Snippet: Driver mutation distribution in patients with lung cancer patients. (A) Plasma ctDNA. (B) FFPE gDNA. ctDNA, circulating tumour DNA; gDNA, genomic DNA.

    Techniques Used: Mutagenesis, Formalin-fixed Paraffin-Embedded

    41) Product Images from "Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis"

    Article Title: Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1184799

    Inflammation and Th1 skewing in the colon of Atg7 cKO mice. (A) Colitis score was calculated on d 7 of DSS treatment as described in Materials and Methods (n = 5 each) (right). Representative H E sections are shown (left, scale bar: 200 μm) (asterisk, inflammation; arrow, ulceration; arrow head, regeneration; white arrow, transmural lesion). (B) Relative expression of cytokines and chemokines in the colonic tissues on d 7 of DSS treatment assessed by quantitative RT-PCR analysis of mRNA prepared using a formalin-fixed paraffin-embedded extraction kit (n = 8 to 10). (C) Th skewing in the colonic lamina propria on day 7 of DSS treatment was evaluated by flow cytometry. The proportions of IFNG + cells and IL17A + cells among CD3E + CD4 + T cells (lower) (n = 4 each). Representative scattergrams are shown (upper). *, P
    Figure Legend Snippet: Inflammation and Th1 skewing in the colon of Atg7 cKO mice. (A) Colitis score was calculated on d 7 of DSS treatment as described in Materials and Methods (n = 5 each) (right). Representative H E sections are shown (left, scale bar: 200 μm) (asterisk, inflammation; arrow, ulceration; arrow head, regeneration; white arrow, transmural lesion). (B) Relative expression of cytokines and chemokines in the colonic tissues on d 7 of DSS treatment assessed by quantitative RT-PCR analysis of mRNA prepared using a formalin-fixed paraffin-embedded extraction kit (n = 8 to 10). (C) Th skewing in the colonic lamina propria on day 7 of DSS treatment was evaluated by flow cytometry. The proportions of IFNG + cells and IL17A + cells among CD3E + CD4 + T cells (lower) (n = 4 each). Representative scattergrams are shown (upper). *, P

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded, Flow Cytometry, Cytometry

    42) Product Images from "Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent"

    Article Title: Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.14741.2

    DNA methylation across the body of the HTLV-1 provirus. ( a ) Upper panel: count of CpG dinucleotides in a window of 350 bp in the HTLV-1 reference genome (L36905). Lower panel: schematic diagram of HTLV-1 provirus indicating the two LTRs and the 9 loci examined by MeDIP. ( b ) DNA methylation on the HTLV-1 provirus in the Tax + and Tax – populations from two HTLV-1-infected T cell clones (Clones TBX4B and 11.65). ( c ) DNA methylation on the HTLV-1 provirus in the CADM1 + Tax + and CADM1 + Tax – populations in PBMCs from two unrelated individuals (Patients TDZ and TED). The asterisk (*) indicates that the PCR failed to amplify.
    Figure Legend Snippet: DNA methylation across the body of the HTLV-1 provirus. ( a ) Upper panel: count of CpG dinucleotides in a window of 350 bp in the HTLV-1 reference genome (L36905). Lower panel: schematic diagram of HTLV-1 provirus indicating the two LTRs and the 9 loci examined by MeDIP. ( b ) DNA methylation on the HTLV-1 provirus in the Tax + and Tax – populations from two HTLV-1-infected T cell clones (Clones TBX4B and 11.65). ( c ) DNA methylation on the HTLV-1 provirus in the CADM1 + Tax + and CADM1 + Tax – populations in PBMCs from two unrelated individuals (Patients TDZ and TED). The asterisk (*) indicates that the PCR failed to amplify.

    Techniques Used: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Infection, Clone Assay, Polymerase Chain Reaction

    DNA methylation in the HTLV-1 LTR of patient-derived PBMCs. ( a ) The HTLV-1 LTR sequence (Accession no. L36905) with CpG dinucleotides highlighted in bold. The three TREs are coloured in red; the TATA box is indicated in the rectangle. ( b ) Schematic diagram of HTLV-1 provirus and the regions amplified with indicated sets of primers for bisulfite-sequencing. Sequencing results for each region are shown in the corresponding panels ( c – f ). The three TREs are indicated by red bars. ( c – f ) Schematic representation of DNA methylation for each clone sequenced. Open circles indicate unmethylated cytosine; closed circles methylated cytosine. The numbers indicate the corresponding CpG sites in panel ( a ).
    Figure Legend Snippet: DNA methylation in the HTLV-1 LTR of patient-derived PBMCs. ( a ) The HTLV-1 LTR sequence (Accession no. L36905) with CpG dinucleotides highlighted in bold. The three TREs are coloured in red; the TATA box is indicated in the rectangle. ( b ) Schematic diagram of HTLV-1 provirus and the regions amplified with indicated sets of primers for bisulfite-sequencing. Sequencing results for each region are shown in the corresponding panels ( c – f ). The three TREs are indicated by red bars. ( c – f ) Schematic representation of DNA methylation for each clone sequenced. Open circles indicate unmethylated cytosine; closed circles methylated cytosine. The numbers indicate the corresponding CpG sites in panel ( a ).

    Techniques Used: DNA Methylation Assay, Derivative Assay, Sequencing, Amplification, Methylation Sequencing, Methylation

    Epigenetic modifications in the HTLV-1 provirus lacking CTCF binding. ( a ) Histone modifications in the Tax + and Tax – populations of the altered HTLV-1-infected T cell clone (Subclone #78 of TBX4B). ( b ) DNA methylation in the body of the provirus in TBX4B-78. Note the similarity to the profiles of epigenetic modifications in the wild-type TBX4B ( Figure 3 ).
    Figure Legend Snippet: Epigenetic modifications in the HTLV-1 provirus lacking CTCF binding. ( a ) Histone modifications in the Tax + and Tax – populations of the altered HTLV-1-infected T cell clone (Subclone #78 of TBX4B). ( b ) DNA methylation in the body of the provirus in TBX4B-78. Note the similarity to the profiles of epigenetic modifications in the wild-type TBX4B ( Figure 3 ).

    Techniques Used: Binding Assay, Infection, DNA Methylation Assay

    43) Product Images from "Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium"

    Article Title: Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium

    Journal: Cell Stem Cell

    doi: 10.1016/j.stem.2018.04.020

    Expansion Coefficient Predicts Age-Related Mutation Burden (A) Simulation demonstrating mutation rate determines accumulated mutation burden at age 60 years for neutral genes. (B) Simulated mutation burden of the colon plotted against patient age for notional genes sharing a common mutation rate (2 × 10 −6 /mitosis). Plots show neutral outcome (green), mutation conferring increased P R (0.99) only (blue), mutation conferring 3-fold increase in rate of fission only (purple), and mutations conferring both increased 3-fold fission rate and P R of 0.99 (red) that corresponds to the observed consequence of STAG2 mutation. (C) Mutant allele frequency data of KRAS (G12D) mutations from 20 patients determined using allele-specific competitive blocker (ACB)-PCR method. Patient data are represented by red circles. The mean (black line) and 95% CI (grayed area) of the model is shown. Red dotted line shows detection threshold. Inset demonstrating the contribution of fission shows the predicted average accumulation of KRAS (G12D) mutant alleles with inferred elevated (black) and wild-type (blue) fission rates, respectively. (D) Boxplot to show the accumulation of KRAS mutant crypts using both the ACB-PCR method and targeted amplicon sequencing on a separate set of patients. > 95% ME. (E) Boxplot to show percentage of KRAS mutant crypts undergoing fission per year using both the ACB-PCR and targeted amplicon data. > 95% ME. (F) Mutant allele frequency data of KRAS mutations from 126 individuals plotted against age. 13 individuals displayed detectable mutations, and the mean accumulation of mutant allele calculated using the model is plotted (black line) as well as the 95% CI. Red dotted line shows detection threshold. (G) The calculated patch size of crypts mutant in respect of KRAS , STAG2 , or MAOA /mPAS shows a significant expansion of KRAS mutant patches in the human colon following clone fixation. (H) The average patch size of each clonal mark plotted against the number of fixed clones per colon shows a small number of small clones for MAOA, with similar patch sizes but higher in frequency for mPAS and STAG2. While for KRAS a small number of large patches is predicted. (I) Lifetime coefficient of expansion normalized to a neutral mark (mPAS shown) allows comparison of relative advantage. See also Figure S4 .
    Figure Legend Snippet: Expansion Coefficient Predicts Age-Related Mutation Burden (A) Simulation demonstrating mutation rate determines accumulated mutation burden at age 60 years for neutral genes. (B) Simulated mutation burden of the colon plotted against patient age for notional genes sharing a common mutation rate (2 × 10 −6 /mitosis). Plots show neutral outcome (green), mutation conferring increased P R (0.99) only (blue), mutation conferring 3-fold increase in rate of fission only (purple), and mutations conferring both increased 3-fold fission rate and P R of 0.99 (red) that corresponds to the observed consequence of STAG2 mutation. (C) Mutant allele frequency data of KRAS (G12D) mutations from 20 patients determined using allele-specific competitive blocker (ACB)-PCR method. Patient data are represented by red circles. The mean (black line) and 95% CI (grayed area) of the model is shown. Red dotted line shows detection threshold. Inset demonstrating the contribution of fission shows the predicted average accumulation of KRAS (G12D) mutant alleles with inferred elevated (black) and wild-type (blue) fission rates, respectively. (D) Boxplot to show the accumulation of KRAS mutant crypts using both the ACB-PCR method and targeted amplicon sequencing on a separate set of patients. > 95% ME. (E) Boxplot to show percentage of KRAS mutant crypts undergoing fission per year using both the ACB-PCR and targeted amplicon data. > 95% ME. (F) Mutant allele frequency data of KRAS mutations from 126 individuals plotted against age. 13 individuals displayed detectable mutations, and the mean accumulation of mutant allele calculated using the model is plotted (black line) as well as the 95% CI. Red dotted line shows detection threshold. (G) The calculated patch size of crypts mutant in respect of KRAS , STAG2 , or MAOA /mPAS shows a significant expansion of KRAS mutant patches in the human colon following clone fixation. (H) The average patch size of each clonal mark plotted against the number of fixed clones per colon shows a small number of small clones for MAOA, with similar patch sizes but higher in frequency for mPAS and STAG2. While for KRAS a small number of large patches is predicted. (I) Lifetime coefficient of expansion normalized to a neutral mark (mPAS shown) allows comparison of relative advantage. See also Figure S4 .

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay

    44) Product Images from "Loss of expression and prognosis value of alpha-internexin in gastroenteropancreatic neuroendocrine neoplasm"

    Article Title: Loss of expression and prognosis value of alpha-internexin in gastroenteropancreatic neuroendocrine neoplasm

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4449-8

    Kaplan-Meier survival curves of patients with GEP-NENs and subtypes of GEP-NENs according to α-internexin expression. a Overall survival by α-internexin expression in GEP-NENs. b Overall survival by α-internexin expression in GI-NENs. c Overall survival by α-internexin expression in pNENs. GEP-NEN: Gastroenteropancreatic neuroendocrine neoplasm; GI-NEN: Gastrointestinal neuroendocrine neoplasm; pNEN: Pancreatic neuroendocrine neoplasm
    Figure Legend Snippet: Kaplan-Meier survival curves of patients with GEP-NENs and subtypes of GEP-NENs according to α-internexin expression. a Overall survival by α-internexin expression in GEP-NENs. b Overall survival by α-internexin expression in GI-NENs. c Overall survival by α-internexin expression in pNENs. GEP-NEN: Gastroenteropancreatic neuroendocrine neoplasm; GI-NEN: Gastrointestinal neuroendocrine neoplasm; pNEN: Pancreatic neuroendocrine neoplasm

    Techniques Used: Expressing

    Correlation of α-internexin methylation with overall survival. a 1, b 1, c 1 Receiver operating characteristic (ROC) curve was used to determine a best cutoff value to define the methylation status of α-internexin in GEP-NENs, subtype of GI-NENs and pNENs. a 2, b 2, c 2 Overall survival by α-internexin methylation (results were examined by methylation level of the average of total 12 CpG sites) in GEP-NENs, subtype of GI-NENs and pNENs. ROC: Receiver operating characteristic; AUC: Area under ROC curve; GEP-NEN: Gastroenteropancreatic neuroendocrine neoplasm; GI-NEN: Gastrointestinal neuroendocrine neoplasm; pNEN: Pancreatic neuroendocrine neoplasm
    Figure Legend Snippet: Correlation of α-internexin methylation with overall survival. a 1, b 1, c 1 Receiver operating characteristic (ROC) curve was used to determine a best cutoff value to define the methylation status of α-internexin in GEP-NENs, subtype of GI-NENs and pNENs. a 2, b 2, c 2 Overall survival by α-internexin methylation (results were examined by methylation level of the average of total 12 CpG sites) in GEP-NENs, subtype of GI-NENs and pNENs. ROC: Receiver operating characteristic; AUC: Area under ROC curve; GEP-NEN: Gastroenteropancreatic neuroendocrine neoplasm; GI-NEN: Gastrointestinal neuroendocrine neoplasm; pNEN: Pancreatic neuroendocrine neoplasm

    Techniques Used: Methylation

    45) Product Images from "Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens"

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034683

    MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.
    Figure Legend Snippet: Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Cell Culture, RNA Extraction, Isolation, Agarose Gel Electrophoresis

    DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.
    Figure Legend Snippet: DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Extraction, Isolation

    Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.
    Figure Legend Snippet: Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Techniques Used: Methylation, Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Isolation, DNA Methylation Assay

    Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.
    Figure Legend Snippet: Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Electrophoresis

    Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: RNA Expression, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    46) Product Images from "Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel"

    Article Title: Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-014-0062-0

    Technology overview of NGS using the 1052-amplicon targeted sequencing panel. Sample qualification is performed using a real-time PCR assay to determine the percent “functional” DNA copies in FFPE samples using the Quantitative Functional Index (QFI). The library preparation is based on RainDance technologies picodroplet PCR to amplify target sequences with subsequent sequencing on an Illumina platform (GAIIx or HiSeq). The bioinformatics workflow trims adaptors and filters out low-quality reads, then includes genomic alignments, variant calling and result reporting (See Materials and Methods for more details and Additional file 1 : Figure S6 for the result visualization).
    Figure Legend Snippet: Technology overview of NGS using the 1052-amplicon targeted sequencing panel. Sample qualification is performed using a real-time PCR assay to determine the percent “functional” DNA copies in FFPE samples using the Quantitative Functional Index (QFI). The library preparation is based on RainDance technologies picodroplet PCR to amplify target sequences with subsequent sequencing on an Illumina platform (GAIIx or HiSeq). The bioinformatics workflow trims adaptors and filters out low-quality reads, then includes genomic alignments, variant calling and result reporting (See Materials and Methods for more details and Additional file 1 : Figure S6 for the result visualization).

    Techniques Used: Next-Generation Sequencing, Amplification, Sequencing, Real-time Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Functional Assay, Polymerase Chain Reaction, Variant Assay

    The effects of input DNA quality and quantity on variant detection background distributions. A) The figure shows variant calling background for the indicated mass amounts of intact cell line or comprised quality, FFPE sample DNA. The standard deviation of the heterozygous variants increases only slightly with reduced DNA input quantities with intact DNA (left panel), but increases dramatically when the quantity of lower quality, FFPE DNA is reduced (right panel). The 99th percentile of the background percent variant (red and blue lines) is more consistent for cell line DNA than for FFPE DNA with the rise in the background being largely driven by the GC > AT transitions. B) The median 99th percentile of the background for all possible substitutions (with G > A and C > T collapsed into GC > AT) from TAS analysis using 250 ng DNA from 8 FFPE samples using the 1052-amplicon panel (x-axis) compared to after target enrichment using an independent 35-amplicon panel (y-axis). As expected, GC > AT transitions contribute higher background than other possible substitutions.
    Figure Legend Snippet: The effects of input DNA quality and quantity on variant detection background distributions. A) The figure shows variant calling background for the indicated mass amounts of intact cell line or comprised quality, FFPE sample DNA. The standard deviation of the heterozygous variants increases only slightly with reduced DNA input quantities with intact DNA (left panel), but increases dramatically when the quantity of lower quality, FFPE DNA is reduced (right panel). The 99th percentile of the background percent variant (red and blue lines) is more consistent for cell line DNA than for FFPE DNA with the rise in the background being largely driven by the GC > AT transitions. B) The median 99th percentile of the background for all possible substitutions (with G > A and C > T collapsed into GC > AT) from TAS analysis using 250 ng DNA from 8 FFPE samples using the 1052-amplicon panel (x-axis) compared to after target enrichment using an independent 35-amplicon panel (y-axis). As expected, GC > AT transitions contribute higher background than other possible substitutions.

    Techniques Used: Variant Assay, Formalin-fixed Paraffin-Embedded, Standard Deviation, Amplification

    Interplatform variant dection concordance stratified by QFI. DNA from 72 thyroid cancer FFPE samples was used to test the concordance of variant calling results between 1052-amplicon panel and a liquid bead array system (Luminex). The samples with the highest quality DNA (QFI > 3%) showed the strongest concordance, > 99% between the two variant detection platforms. The concordance rate decreases with reduced QFI. The size of the markers is based on Luminex status (large markers are mutants, small markers are wild type). The color of the markers is based on the classification status of the 1052-amplicon panel with data from the Luminex platform defined as truth: green is for true positives (TP); gray is for true negatives (TN); orange is for false positives (FP); red is for false negatives (FN); and black is for low sequencing coverage by the 1052-amplicon panel (Low Coverage). The specific predictions from the 1052-amplicon panel are defined as wild type (circles), mutant (triangle) and low coverage (square). The number of rows in the agreement maps corresponds to the number of samples in the study except when a sample had no coverage at any of the 15 specified hypotheses. For example, the left most panel has 18 rows corresponding to all 18 samples that were sequenced (with a total of 15 hypotheses *18 samples = 270 entries) so the 4x2 table above the agreement map sums to 270. However, the right most panel only has 14 rows representing 26 samples. That is, 12 samples had no coverage associated with the 15 hypotheses and are not shown, but the corresponding 4x2 table still sums to 15 hypotheses *26 samples = 390 entries. Incidentally, none of the samples that had zero coverage by the 1052-amplicon panel workflow had positive calls using the Luminex method.
    Figure Legend Snippet: Interplatform variant dection concordance stratified by QFI. DNA from 72 thyroid cancer FFPE samples was used to test the concordance of variant calling results between 1052-amplicon panel and a liquid bead array system (Luminex). The samples with the highest quality DNA (QFI > 3%) showed the strongest concordance, > 99% between the two variant detection platforms. The concordance rate decreases with reduced QFI. The size of the markers is based on Luminex status (large markers are mutants, small markers are wild type). The color of the markers is based on the classification status of the 1052-amplicon panel with data from the Luminex platform defined as truth: green is for true positives (TP); gray is for true negatives (TN); orange is for false positives (FP); red is for false negatives (FN); and black is for low sequencing coverage by the 1052-amplicon panel (Low Coverage). The specific predictions from the 1052-amplicon panel are defined as wild type (circles), mutant (triangle) and low coverage (square). The number of rows in the agreement maps corresponds to the number of samples in the study except when a sample had no coverage at any of the 15 specified hypotheses. For example, the left most panel has 18 rows corresponding to all 18 samples that were sequenced (with a total of 15 hypotheses *18 samples = 270 entries) so the 4x2 table above the agreement map sums to 270. However, the right most panel only has 14 rows representing 26 samples. That is, 12 samples had no coverage associated with the 15 hypotheses and are not shown, but the corresponding 4x2 table still sums to 15 hypotheses *26 samples = 390 entries. Incidentally, none of the samples that had zero coverage by the 1052-amplicon panel workflow had positive calls using the Luminex method.

    Techniques Used: Variant Assay, Formalin-fixed Paraffin-Embedded, Amplification, Luminex, Sequencing, Mutagenesis

    47) Product Images from "A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens"

    Article Title: A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161274

    Multiplex (4-plex) ddPCR output generated using either a standard orthogonal layout of droplet clusters or our staggered layout technique. Standard orthogonal layout of droplet data for DNA extracted from ( A ) normal blood or ( B ) normal FFPE tissue. Staggered layout of droplet data for DNA extracted from ( C ) normal blood or ( D ) normal FFPE tissue. The droplets shown in each plot are a combination of n = 4 replicates and the four primary clusters are labelled. The four loci amplified were 1 = ACADM ; 2 = KCNS3 ; 3 = SLC25A12 and 4 = HFE2 . The orthogonal layout was achieved using the following concentrations of labelled probes (1 = 600 nM FAM; 2 = 300 nM FAM; 3 = 300 nM HEX; 4 = 600 nM HEX). The staggered layout was achieved using the recipe described in main text. Each plot was created by the overlayed output data for a set of n = 4 multiplex ddPCR experiments.
    Figure Legend Snippet: Multiplex (4-plex) ddPCR output generated using either a standard orthogonal layout of droplet clusters or our staggered layout technique. Standard orthogonal layout of droplet data for DNA extracted from ( A ) normal blood or ( B ) normal FFPE tissue. Staggered layout of droplet data for DNA extracted from ( C ) normal blood or ( D ) normal FFPE tissue. The droplets shown in each plot are a combination of n = 4 replicates and the four primary clusters are labelled. The four loci amplified were 1 = ACADM ; 2 = KCNS3 ; 3 = SLC25A12 and 4 = HFE2 . The orthogonal layout was achieved using the following concentrations of labelled probes (1 = 600 nM FAM; 2 = 300 nM FAM; 3 = 300 nM HEX; 4 = 600 nM HEX). The staggered layout was achieved using the recipe described in main text. Each plot was created by the overlayed output data for a set of n = 4 multiplex ddPCR experiments.

    Techniques Used: Multiplex Assay, Generated, Formalin-fixed Paraffin-Embedded, Amplification

    Analysis of DNA fragmentation as a function of template length and type of sample. ln ( R i/cr ) M values determined from triplex ddPCR experiments on gDNA recovered from normal blood (open symbols) or FFPE tissue specimens (filled symbols). Each triplex experiment co-amplifies the HER2 cr and one length i each of the CPT2 length-based control (circles) and the KCNS3 length-based control (triangles). Error bars represent a 95% confidence interval.
    Figure Legend Snippet: Analysis of DNA fragmentation as a function of template length and type of sample. ln ( R i/cr ) M values determined from triplex ddPCR experiments on gDNA recovered from normal blood (open symbols) or FFPE tissue specimens (filled symbols). Each triplex experiment co-amplifies the HER2 cr and one length i each of the CPT2 length-based control (circles) and the KCNS3 length-based control (triangles). Error bars represent a 95% confidence interval.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    48) Product Images from "Fit for genomic and proteomic purposes: Sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue"

    Article Title: Fit for genomic and proteomic purposes: Sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181756

    Box and whiskers plots showing the distribution of absorbance ratios (A 260/280 ) for nucleic acids DNA, RNA, miRNA and for protein (A 280 ) from FFPE. Panel A shows derivatives from FFPE stored between 1990–2001 and Panel B derivative from FFPE stored between 2002–2013 stratified by storage duration (11 year intervals) and cancer tumor tissue type (adenocarcinoma, squamous cell, and papillary carcinoma).
    Figure Legend Snippet: Box and whiskers plots showing the distribution of absorbance ratios (A 260/280 ) for nucleic acids DNA, RNA, miRNA and for protein (A 280 ) from FFPE. Panel A shows derivatives from FFPE stored between 1990–2001 and Panel B derivative from FFPE stored between 2002–2013 stratified by storage duration (11 year intervals) and cancer tumor tissue type (adenocarcinoma, squamous cell, and papillary carcinoma).

    Techniques Used: Formalin-fixed Paraffin-Embedded

    Fit for genomic and proteomic purposes by steps (numbered yellow boxes). In step 1, the ACCESS database of the ACSR at GWU was used to construct a sampling frame of available FFPE blocks by cancer case, which were the sampling units used to avoid selecting multiple FFPE blocks from the same individual. In step 2, the sampling frame of FFPE blocks was stratified by intervals of 11 years of storage (1990–2001 and 2002–2013, inclusive) and then the three tumor tissue types with the highest frequency of FFPE in the ACSR at GWU. Simple random sampling without replacement was conducted in each stratum until the targeted sample size of 20 FFPE blocks per storage duration and tumor tissue type was reached. In step 3 , commercial kits were employed to extract nucleic acids and protein from 10 μm FFPE sections from each block; a separate FFPE section was used for each type of nucleic acid or protein extraction. In step 4, an initial assessment for the presence of the nucleic acid or protein was conducted by ultraviolet absorbance (UV). In step 5, the purity and concentration of nucleic acid and protein extracts were determined by a SpectraDrop Micro-Volume Microplate with a SPECTRAmax 384PLUS plate reader and SoftMax Pro v6.4.1 software for device control and data analysis. In step 6, nucleic acid and protein derivatives were assigned to fitness categories as described in the Methods section. In step 7 , the fitness of each FFPE block was assessed by ranking the combined fitness of the derivatives as follows: FFPE blocks that met the “Fit Nucleic Acids and Proteins for Diverse Analyses” requirements included blocks in which all four derivatives (DNA, RNA, miRNA, and protein) were “Fit”. FFPE blocks that were categorized as “Fit Nucleic Acids for Diverse Genomic Analyses” included blocks that were determined to have “Fit” nucleic acid derivatives only. FFPE blocks that had one or two
    Figure Legend Snippet: Fit for genomic and proteomic purposes by steps (numbered yellow boxes). In step 1, the ACCESS database of the ACSR at GWU was used to construct a sampling frame of available FFPE blocks by cancer case, which were the sampling units used to avoid selecting multiple FFPE blocks from the same individual. In step 2, the sampling frame of FFPE blocks was stratified by intervals of 11 years of storage (1990–2001 and 2002–2013, inclusive) and then the three tumor tissue types with the highest frequency of FFPE in the ACSR at GWU. Simple random sampling without replacement was conducted in each stratum until the targeted sample size of 20 FFPE blocks per storage duration and tumor tissue type was reached. In step 3 , commercial kits were employed to extract nucleic acids and protein from 10 μm FFPE sections from each block; a separate FFPE section was used for each type of nucleic acid or protein extraction. In step 4, an initial assessment for the presence of the nucleic acid or protein was conducted by ultraviolet absorbance (UV). In step 5, the purity and concentration of nucleic acid and protein extracts were determined by a SpectraDrop Micro-Volume Microplate with a SPECTRAmax 384PLUS plate reader and SoftMax Pro v6.4.1 software for device control and data analysis. In step 6, nucleic acid and protein derivatives were assigned to fitness categories as described in the Methods section. In step 7 , the fitness of each FFPE block was assessed by ranking the combined fitness of the derivatives as follows: FFPE blocks that met the “Fit Nucleic Acids and Proteins for Diverse Analyses” requirements included blocks in which all four derivatives (DNA, RNA, miRNA, and protein) were “Fit”. FFPE blocks that were categorized as “Fit Nucleic Acids for Diverse Genomic Analyses” included blocks that were determined to have “Fit” nucleic acid derivatives only. FFPE blocks that had one or two "fit" or "above fit" derivative out of the three nucleic acid derivatives and “unfit”, "fit" or "above fit" protein derivatives, were considered “Fit for a Specific Genomic or Proteomic Analysis”. In step 8, if an FFPE block had no “Fit” molecular derivatives, it was considered a “Bad Block”.

    Techniques Used: Construct, Sampling, Formalin-fixed Paraffin-Embedded, Blocking Assay, Protein Extraction, Concentration Assay, Software

    Box and whiskers plots showing the distribution of concentration (nanograms per microliter) for the nucleic acids DNA, RNA, and miRNA co-extracted from FFPE. Panel A shows derivatives from FFPE stored between 1990–2001 and Panel B shows derivative from FFPE stored between 2002–2013.
    Figure Legend Snippet: Box and whiskers plots showing the distribution of concentration (nanograms per microliter) for the nucleic acids DNA, RNA, and miRNA co-extracted from FFPE. Panel A shows derivatives from FFPE stored between 1990–2001 and Panel B shows derivative from FFPE stored between 2002–2013.

    Techniques Used: Concentration Assay, Formalin-fixed Paraffin-Embedded

    49) Product Images from "Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method"

    Article Title: Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-12-12

    Pyrograms of the LINE-1, MLH1 and MGMT methylation markers for different couples of frozen/FFPE DNA . Pyrograms of LINE-1 marker are those obtained for couple n° 10 ( A and B ) and for MLH1 and MGMT markers those for couples n°13 ( C and D ) and n°6 ( E and F ) respectively. Arrows indicate positions of internal controls of conversion, demonstrating no residual cytosines at the non-CpG sites. Gray areas indicate polymorphisms, between T/C, generated by bisulfite treatment. Level of methylation for a given CpG dinucleotide is reported above it (gray square).
    Figure Legend Snippet: Pyrograms of the LINE-1, MLH1 and MGMT methylation markers for different couples of frozen/FFPE DNA . Pyrograms of LINE-1 marker are those obtained for couple n° 10 ( A and B ) and for MLH1 and MGMT markers those for couples n°13 ( C and D ) and n°6 ( E and F ) respectively. Arrows indicate positions of internal controls of conversion, demonstrating no residual cytosines at the non-CpG sites. Gray areas indicate polymorphisms, between T/C, generated by bisulfite treatment. Level of methylation for a given CpG dinucleotide is reported above it (gray square).

    Techniques Used: Methylation, Formalin-fixed Paraffin-Embedded, Marker, Generated

    50) Product Images from "Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice"

    Article Title: Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196434

    Deamination events increase with sample age when not treated with UNG. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Deamination events increase with sample age when not treated with UNG. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    Number of deamination variants ranges widely amongst samples when fixation time is uncontrolled. Orange crosses represent number of deamination variants (y-axis; C- > T/G- > A). Blue dots represent all other possible variants. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Number of deamination variants ranges widely amongst samples when fixation time is uncontrolled. Orange crosses represent number of deamination variants (y-axis; C- > T/G- > A). Blue dots represent all other possible variants. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    Read quality in the baseline group does not change by DNA extraction type. Read quality as measured by percent reads mapped. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Read quality in the baseline group does not change by DNA extraction type. Read quality as measured by percent reads mapped. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Total reads mapped in the baseline group does not change by DNA extraction type. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Total reads mapped in the baseline group does not change by DNA extraction type. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Sequencing read quality decreases over fixation time. Read quality as a measure of percent reads mapped (y-axis) decreases with a longer tissue fixation time (x-axis). Blue dots represent DNA without UNG treatment (extracted using QiaAmp FFPE kit; QA), orange crossed represent matched DNA treated with UNG (extraction using the GeneRead kit; GR UNG). Linear regression line depicted by the central broken line and encompassed by dotted lines representing the 95% confidence interval (CI region colored). Frozen samples used for ground truth are represented by green dots. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Sequencing read quality decreases over fixation time. Read quality as a measure of percent reads mapped (y-axis) decreases with a longer tissue fixation time (x-axis). Blue dots represent DNA without UNG treatment (extracted using QiaAmp FFPE kit; QA), orange crossed represent matched DNA treated with UNG (extraction using the GeneRead kit; GR UNG). Linear regression line depicted by the central broken line and encompassed by dotted lines representing the 95% confidence interval (CI region colored). Frozen samples used for ground truth are represented by green dots. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

    Read quality decreases with sample age in the age group. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
    Figure Legend Snippet: Read quality decreases with sample age in the age group. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    51) Product Images from "Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing"

    Article Title: Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep20944

    DEPArray™ analysis and gating based on scatter plots, histogram and images. ( a ) Simultaneous detection of two distinct cell populations based on antigen expression from a dissociated FFPE specimen by using the mean intensity keratin-Alexa488 vs vimentin-Alexa647 scatter plot. ( b ) After gating for the vimentin-positive (V + ) and keratin-positive (K + ) cell populations, single-parameter DNA histograms are generated. The V + fraction is used as an internal DNA-diploid reference for K + population DNA content assessment. ( c ) Images of individual events can be viewed on an image bar, allowing cells of interest to be identified and flagged for recovery.
    Figure Legend Snippet: DEPArray™ analysis and gating based on scatter plots, histogram and images. ( a ) Simultaneous detection of two distinct cell populations based on antigen expression from a dissociated FFPE specimen by using the mean intensity keratin-Alexa488 vs vimentin-Alexa647 scatter plot. ( b ) After gating for the vimentin-positive (V + ) and keratin-positive (K + ) cell populations, single-parameter DNA histograms are generated. The V + fraction is used as an internal DNA-diploid reference for K + population DNA content assessment. ( c ) Images of individual events can be viewed on an image bar, allowing cells of interest to be identified and flagged for recovery.

    Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded, Generated

    DNA content categories from cells suspension derived from FFPE lung tumor based on multi-parametric analysis. ( a,b ) DNA content histogram show a DNA diploid peak after gating of the V + K cell fraction. K + V- population shows a DNA histogram containing two DNA fractions: a DNA diploid fraction with a DNA index of 0.97 and an hyperdiploid fraction with a DNA index of 1.56. ( c ) DNA-content distribution of cell recoveries. The dots with the same color represent an homogenous cell type, green (K + V- DI = 0.97), dark-green (K + V- DI = 1.56) and red (V + K- DNA diploid). ( d ) Sample S09 frequency profiles show different mutational profiles between stromal, DNA diploid and DNA aneuploid tumor populations. Across the loci displayed (one SNP and two somatic mutations), the DNA aneuploid tumor has a much higher frequency value than the diploid tumor population. SG = stop_gain, M = missense. The variants of the diploid K + V- population (DI = 0,96) confirm its tumor origin.
    Figure Legend Snippet: DNA content categories from cells suspension derived from FFPE lung tumor based on multi-parametric analysis. ( a,b ) DNA content histogram show a DNA diploid peak after gating of the V + K cell fraction. K + V- population shows a DNA histogram containing two DNA fractions: a DNA diploid fraction with a DNA index of 0.97 and an hyperdiploid fraction with a DNA index of 1.56. ( c ) DNA-content distribution of cell recoveries. The dots with the same color represent an homogenous cell type, green (K + V- DI = 0.97), dark-green (K + V- DI = 1.56) and red (V + K- DNA diploid). ( d ) Sample S09 frequency profiles show different mutational profiles between stromal, DNA diploid and DNA aneuploid tumor populations. Across the loci displayed (one SNP and two somatic mutations), the DNA aneuploid tumor has a much higher frequency value than the diploid tumor population. SG = stop_gain, M = missense. The variants of the diploid K + V- population (DI = 0,96) confirm its tumor origin.

    Techniques Used: Derivative Assay, Formalin-fixed Paraffin-Embedded

    52) Product Images from "Systematic evaluation of PAXgene® tissue fixation for the histopathological and molecular study of lung cancer"

    Article Title: Systematic evaluation of PAXgene® tissue fixation for the histopathological and molecular study of lung cancer

    Journal: The Journal of Pathology: Clinical Research

    doi: 10.1002/cjp2.145

    DNA analysis. DNA concentrations were greater in (A) PFPE lung parenchyma (4714 ± 839.3 ng versus 583.4 ± 125.9 ng, p = 0.0011), (B) PFPE tumour blocks, (9476 ng ±1371 ng versus 1955 ng ± 304.6 ng, p = 0.008) and (C) PFPE Temno biopsies (770.1 ± 181.7 ng versus 42.5 ± 7.44, p = 0.0053), than FFPE comparators. RNA RT PCR mean C T values ( n = 8) for β‐actin were lower (D) in PFPE lung parenchyma (31.48 ± 0.8484 versus 38.86 ± 0.6143, p = 0.002), block‐sized tumour samples (26.69 ± 0.2638 versus 38.01 ± 0.8508, p
    Figure Legend Snippet: DNA analysis. DNA concentrations were greater in (A) PFPE lung parenchyma (4714 ± 839.3 ng versus 583.4 ± 125.9 ng, p = 0.0011), (B) PFPE tumour blocks, (9476 ng ±1371 ng versus 1955 ng ± 304.6 ng, p = 0.008) and (C) PFPE Temno biopsies (770.1 ± 181.7 ng versus 42.5 ± 7.44, p = 0.0053), than FFPE comparators. RNA RT PCR mean C T values ( n = 8) for β‐actin were lower (D) in PFPE lung parenchyma (31.48 ± 0.8484 versus 38.86 ± 0.6143, p = 0.002), block‐sized tumour samples (26.69 ± 0.2638 versus 38.01 ± 0.8508, p

    Techniques Used: Formalin-fixed Paraffin-Embedded, Reverse Transcription Polymerase Chain Reaction, Blocking Assay

    53) Product Images from "Expanding epigenomics to archived FFPE tissues: An evaluation of DNA repair methodologies"

    Article Title: Expanding epigenomics to archived FFPE tissues: An evaluation of DNA repair methodologies

    Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

    doi: 10.1158/1055-9965.EPI-14-0464

    Schematic of Experimental Parameters comparing FFPE DNA repair methods to FF DNA DNA (500ng) from FF tissue was processed according to Illumina Human Methylation instructions, including bisulfite modification followed by the standard Human Methylation processing protocol (Gold standard). Experimental conditions are separated for REPLI-g ligation (LIG) and Illumina Restore Kit (RES). LIG1 : the original Thirwell method using 500ng of genomic DNA processed by REPLI-g ligase and bisulfite modified (BS), and 4µl of bisulfite-modified DNA used for the starting material for the Illumina Human Methylation array kit. LIG2 : Thirwell method with output DNA increased to 8µl of bisulfite modified DNA, which is the same as used in the Restore Kit. LIG3 and LIG4 : 500ng and 250ng of genomic DNA, respectively, were bisulfite modified, processed by REPLI-g ligase and 8µl of material used for Illumina Methylation Array kit. RES1 : the Illumina Restore protocol using 500ng of genomic DNA, bisulfite modified and processed per Restore Kit protocol (including 8µl for Array steps). RES2 and RES3 : Technical replicates for Restore Kit using 250ng of genomic DNA.
    Figure Legend Snippet: Schematic of Experimental Parameters comparing FFPE DNA repair methods to FF DNA DNA (500ng) from FF tissue was processed according to Illumina Human Methylation instructions, including bisulfite modification followed by the standard Human Methylation processing protocol (Gold standard). Experimental conditions are separated for REPLI-g ligation (LIG) and Illumina Restore Kit (RES). LIG1 : the original Thirwell method using 500ng of genomic DNA processed by REPLI-g ligase and bisulfite modified (BS), and 4µl of bisulfite-modified DNA used for the starting material for the Illumina Human Methylation array kit. LIG2 : Thirwell method with output DNA increased to 8µl of bisulfite modified DNA, which is the same as used in the Restore Kit. LIG3 and LIG4 : 500ng and 250ng of genomic DNA, respectively, were bisulfite modified, processed by REPLI-g ligase and 8µl of material used for Illumina Methylation Array kit. RES1 : the Illumina Restore protocol using 500ng of genomic DNA, bisulfite modified and processed per Restore Kit protocol (including 8µl for Array steps). RES2 and RES3 : Technical replicates for Restore Kit using 250ng of genomic DNA.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Methylation, Modification, Ligation

    54) Product Images from "Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens"

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034683

    MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.
    Figure Legend Snippet: Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Cell Culture, RNA Extraction, Isolation, Agarose Gel Electrophoresis

    DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.
    Figure Legend Snippet: DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Extraction, Isolation

    Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.
    Figure Legend Snippet: Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Techniques Used: Methylation, Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Isolation, DNA Methylation Assay

    Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.
    Figure Legend Snippet: Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Electrophoresis

    Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: RNA Expression, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    55) Product Images from "A simple and robust real-time qPCR method for the detection of PIK3CA mutations"

    Article Title: A simple and robust real-time qPCR method for the detection of PIK3CA mutations

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22473-9

    PIK3CA H1047R mutation status analysis in cancer biopsies and FFPE samples. ( A , B ) 10 ng of genomic DNA isolated from frozen core biopsies of a total of 22 cancer patients ( A ) or 10 ng of genomic DNA extracted from FFPE sections of 8 patients ( B) were subjected to qPCR analysis for PIK3CA H1047R mutation. Each assay gave significantly elevated results in the same two samples (patients B12 and B16) in both assays. Data are shown as mean mutant fold change amplification relative to internal control amplification ±SEM. A sample containing a pool of DNA obtained from cell lines carrying the mutation was included in the assay as a positive control. All the experimental points were obtained in triplicates in three independent experiments (n = 3). ***P
    Figure Legend Snippet: PIK3CA H1047R mutation status analysis in cancer biopsies and FFPE samples. ( A , B ) 10 ng of genomic DNA isolated from frozen core biopsies of a total of 22 cancer patients ( A ) or 10 ng of genomic DNA extracted from FFPE sections of 8 patients ( B) were subjected to qPCR analysis for PIK3CA H1047R mutation. Each assay gave significantly elevated results in the same two samples (patients B12 and B16) in both assays. Data are shown as mean mutant fold change amplification relative to internal control amplification ±SEM. A sample containing a pool of DNA obtained from cell lines carrying the mutation was included in the assay as a positive control. All the experimental points were obtained in triplicates in three independent experiments (n = 3). ***P

    Techniques Used: Mutagenesis, Formalin-fixed Paraffin-Embedded, Isolation, Real-time Polymerase Chain Reaction, Amplification, Positive Control

    56) Product Images from "Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach"

    Article Title: Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139074

    KRAS mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect KRAS mutant clones. All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the KRAS mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a KRAS mutant DNA species not present in the multiplex are indicated by a yellow dashed square.
    Figure Legend Snippet: KRAS mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect KRAS mutant clones. All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the KRAS mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a KRAS mutant DNA species not present in the multiplex are indicated by a yellow dashed square.

    Techniques Used: Mutagenesis, Formalin-fixed Paraffin-Embedded, Multiplex Assay, Clone Assay

    Correlation of NGS KRAS mutant allele frequency with digital PCR KRAS mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.
    Figure Legend Snippet: Correlation of NGS KRAS mutant allele frequency with digital PCR KRAS mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.

    Techniques Used: Next-Generation Sequencing, Mutagenesis, Digital PCR, Multiplex Assay, Formalin-fixed Paraffin-Embedded

    Correlation of mutant DNA fraction detected in multiplex and duplex assays using cell line gDNA or oligonucleotides (left) and in FFPE tissue DNA (right).
    Figure Legend Snippet: Correlation of mutant DNA fraction detected in multiplex and duplex assays using cell line gDNA or oligonucleotides (left) and in FFPE tissue DNA (right).

    Techniques Used: Mutagenesis, Multiplex Assay, Formalin-fixed Paraffin-Embedded

    57) Product Images from "High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods"

    Article Title: High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0591-4

    Targeted NGS to detect DNA rearrangements from clincal specimens. ( a ) 7 FFPE resection specimens with ALK fusions identified by IHC were further analyzed by targeted NGS. The results were shown on the table. ALK fusion in sample 3D-65 was further confirmed by FISH ( b ). ( c ) 11 lung adenocarcinoma FFPE specimens that are positive in ALK fusions by targeted NGS were further analyzed by IHC. The results were summarized in the left table. ‘+’ represents positive ALK expression detected by IHC. Representative microscopical results of ALK expression from high to low are shown on the right panel
    Figure Legend Snippet: Targeted NGS to detect DNA rearrangements from clincal specimens. ( a ) 7 FFPE resection specimens with ALK fusions identified by IHC were further analyzed by targeted NGS. The results were shown on the table. ALK fusion in sample 3D-65 was further confirmed by FISH ( b ). ( c ) 11 lung adenocarcinoma FFPE specimens that are positive in ALK fusions by targeted NGS were further analyzed by IHC. The results were summarized in the left table. ‘+’ represents positive ALK expression detected by IHC. Representative microscopical results of ALK expression from high to low are shown on the right panel

    Techniques Used: Next-Generation Sequencing, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Fluorescence In Situ Hybridization, Expressing

    58) Product Images from "Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples"

    Article Title: Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197456

    Variants identified in the samples extracted with different DNA extraction methods. (A-D) Venn diagram showing the distribution of variants between DNA extraction methods in four different FFPE samples, SARC1-4. Variants were detected by MuTect and Strelka, using an artificial control as normal. The data was filtered on exonic variants with allele frequency > 5% and coverage > 100x, being present at
    Figure Legend Snippet: Variants identified in the samples extracted with different DNA extraction methods. (A-D) Venn diagram showing the distribution of variants between DNA extraction methods in four different FFPE samples, SARC1-4. Variants were detected by MuTect and Strelka, using an artificial control as normal. The data was filtered on exonic variants with allele frequency > 5% and coverage > 100x, being present at

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Yield and amplifiability of extracted DNA and RNA. (A) Average total amount of DNA. (B) Average total amount of RNA. (C) Amplifiable DNA quantified with the FFPE QC kit from Illumina. (D) Amplifiable RNA quantified with the PreSeq QC assay from ArcherDx. The average total amount and average delta Ct values for the different samples and extraction methods are shown. The standard deviation is shown as vertical bars. Methods with significant differences in yield are marked as connected with horizontal bars (p
    Figure Legend Snippet: Yield and amplifiability of extracted DNA and RNA. (A) Average total amount of DNA. (B) Average total amount of RNA. (C) Amplifiable DNA quantified with the FFPE QC kit from Illumina. (D) Amplifiable RNA quantified with the PreSeq QC assay from ArcherDx. The average total amount and average delta Ct values for the different samples and extraction methods are shown. The standard deviation is shown as vertical bars. Methods with significant differences in yield are marked as connected with horizontal bars (p

    Techniques Used: Formalin-fixed Paraffin-Embedded, Standard Deviation

    59) Product Images from "Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA"

    Article Title: Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA

    Journal: American Journal of Clinical Pathology

    doi: 10.1093/ajcp/aqw205

    The influence of five different DNA extraction methods on the multiplex ligation-dependent probe amplification (MLPA) probe’s copy number ratios. MLPA was performed on DNA extracted from eight various formalin-fixed, paraffin-embedded (FFPE) tissues with the P027 probe mix (50 probes). The y-axis represents the percentage of probes showing copy number ratios outside the 0.8 to 1.2 normal copy number range. The x-axis shows five different DNA extraction methods. Each bar represents a separate FFPE tissue-type block. For samples where DNA was extracted with the one-tube FFPE extraction method, results from crude (nonpurified) DNA lysate are presented for all tissues, except the lung. The crude lysate of the lung FFPE tissue was also purified. SEM (as indicated by error bars) was calculated by dividing the standard deviation of the mean of the number of probes with copy number ratios outside the normal range by the square root of the number of samples (triplicate) ( Supplementary Table S6 ). colonSymb, tissue from Symbiant Pathology Expert Centre, Alkmaar; colonUMC, tissue from Department of Pathology, University Medical Centre Utrecht, Utrecht.
    Figure Legend Snippet: The influence of five different DNA extraction methods on the multiplex ligation-dependent probe amplification (MLPA) probe’s copy number ratios. MLPA was performed on DNA extracted from eight various formalin-fixed, paraffin-embedded (FFPE) tissues with the P027 probe mix (50 probes). The y-axis represents the percentage of probes showing copy number ratios outside the 0.8 to 1.2 normal copy number range. The x-axis shows five different DNA extraction methods. Each bar represents a separate FFPE tissue-type block. For samples where DNA was extracted with the one-tube FFPE extraction method, results from crude (nonpurified) DNA lysate are presented for all tissues, except the lung. The crude lysate of the lung FFPE tissue was also purified. SEM (as indicated by error bars) was calculated by dividing the standard deviation of the mean of the number of probes with copy number ratios outside the normal range by the square root of the number of samples (triplicate) ( Supplementary Table S6 ). colonSymb, tissue from Symbiant Pathology Expert Centre, Alkmaar; colonUMC, tissue from Department of Pathology, University Medical Centre Utrecht, Utrecht.

    Techniques Used: DNA Extraction, Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification, Formalin-fixed Paraffin-Embedded, Blocking Assay, Purification, Standard Deviation

    (cont) C , MLPA electropherogram of FFPE lung tissue crude lysate column purified with the DNA Clean Concentrator-5 kit. RFU, residual fluorescence unit.
    Figure Legend Snippet: (cont) C , MLPA electropherogram of FFPE lung tissue crude lysate column purified with the DNA Clean Concentrator-5 kit. RFU, residual fluorescence unit.

    Techniques Used: Multiplex Ligation-dependent Probe Amplification, Formalin-fixed Paraffin-Embedded, Purification, Fluorescence

    60) Product Images from "Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells"

    Article Title: Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26495

    Comparison of cancer spheroid- and FFPE tumor-derived DNA samples in sequence analysis of MMR-target coding mononucleotide repeats ( A ) Agarose gel profile comparing the DNA samples from spheroid and FFPE tumors. ( B ) Mutation profiles of coding mononucleotides in four MMR-target genes for seven MSI cases analyzed with spheroid- and FFPE tissue-derived DNA samples. Red boxes/triangles indicate mutated alleles, whereas white ones show the wild-type. Analysis with spheroid-derived DNA enabled unambiguous identification of both alleles. However, FFPE tissue-derived DNA often gave confusing results. ( C , top) An example of homozygous mononucleotide repeat mutation in BAX (G8 → G7), detected using the spheroid-derived DNA. Arrow points 8th G → T (C → A in reverse strand sequenced here). (bottom) With the FFPE tissue-derived DNA, a slightly low peak for the 8th G was detected after seven G peaks (C in reverse strand here). This is likely derived from DNA of the normal (i.e., wild-type) stromal cells rather than cancer epithelial cells.
    Figure Legend Snippet: Comparison of cancer spheroid- and FFPE tumor-derived DNA samples in sequence analysis of MMR-target coding mononucleotide repeats ( A ) Agarose gel profile comparing the DNA samples from spheroid and FFPE tumors. ( B ) Mutation profiles of coding mononucleotides in four MMR-target genes for seven MSI cases analyzed with spheroid- and FFPE tissue-derived DNA samples. Red boxes/triangles indicate mutated alleles, whereas white ones show the wild-type. Analysis with spheroid-derived DNA enabled unambiguous identification of both alleles. However, FFPE tissue-derived DNA often gave confusing results. ( C , top) An example of homozygous mononucleotide repeat mutation in BAX (G8 → G7), detected using the spheroid-derived DNA. Arrow points 8th G → T (C → A in reverse strand sequenced here). (bottom) With the FFPE tissue-derived DNA, a slightly low peak for the 8th G was detected after seven G peaks (C in reverse strand here). This is likely derived from DNA of the normal (i.e., wild-type) stromal cells rather than cancer epithelial cells.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Sequencing, Agarose Gel Electrophoresis, Mutagenesis

    Electropherograms of on-chip MSI analysis using spheroid-derived DNA samples ( A ) HC51T. Representative electropherograms of an MSS case for five microsatellite markers of the Bethesda panel. Red lines show the PCR products amplified from the cancer cell spheroid DNA samples, whereas blue lines indicate those from the normal epithelial stem cell DNA of the same patient. HC26T and HC4T. Representative electropherograms of MSI-H cases. The peak patterns between tumor-initiating cells (red) and the normal epithelial stem cells (blue) are separated for all loci tested. The ordinate shows fluorescence intensity in arbitrary unit (FU). ( B ) On-chip electropherogram of a representative case in which FFPE tumor-derived DNA sample (blue) showed much lower peaks with poorer resolution than spheroid-derived DNA (red) of the same patient normal mucosal stem cells (HC6N). The ordinate shows fluorescence intensity in arbitrary unit (FU). ( C ) Maximum electrophoretic peak-heights of PCR-amplified MSI markers (shown at bottom) compared between spheroid- and FFPE tumor-derived DNA samples. Note that spheroid-derived (Sph) DNA gave taller peaks than FFPE tumor-derived (FFPE) DNA for all five Bethesda panel markers. **** P
    Figure Legend Snippet: Electropherograms of on-chip MSI analysis using spheroid-derived DNA samples ( A ) HC51T. Representative electropherograms of an MSS case for five microsatellite markers of the Bethesda panel. Red lines show the PCR products amplified from the cancer cell spheroid DNA samples, whereas blue lines indicate those from the normal epithelial stem cell DNA of the same patient. HC26T and HC4T. Representative electropherograms of MSI-H cases. The peak patterns between tumor-initiating cells (red) and the normal epithelial stem cells (blue) are separated for all loci tested. The ordinate shows fluorescence intensity in arbitrary unit (FU). ( B ) On-chip electropherogram of a representative case in which FFPE tumor-derived DNA sample (blue) showed much lower peaks with poorer resolution than spheroid-derived DNA (red) of the same patient normal mucosal stem cells (HC6N). The ordinate shows fluorescence intensity in arbitrary unit (FU). ( C ) Maximum electrophoretic peak-heights of PCR-amplified MSI markers (shown at bottom) compared between spheroid- and FFPE tumor-derived DNA samples. Note that spheroid-derived (Sph) DNA gave taller peaks than FFPE tumor-derived (FFPE) DNA for all five Bethesda panel markers. **** P

    Techniques Used: Chromatin Immunoprecipitation, Derivative Assay, Polymerase Chain Reaction, Amplification, Fluorescence, Formalin-fixed Paraffin-Embedded

    Schematic summary of the analysis results using colorectal cancer spheroids compared with those using FFPE tumors ( A ) Mutational burden estimated by exonic sequencing of spheroid DNA for 409 cancer related genes. ( B ) MSI status judged by on-chip electrophoresis of the Bethesda panel markers. ( C ) Mutations in the mononucleotide repeats in coding regions of TGFBR2 and BAX determined using spheroid and FFPE tumor DNA. ( D ) IHC results of the cultured spheroids of tumor-initiating cells and FFPE primary tumors. Color keys are shown in boxes. See text for details.
    Figure Legend Snippet: Schematic summary of the analysis results using colorectal cancer spheroids compared with those using FFPE tumors ( A ) Mutational burden estimated by exonic sequencing of spheroid DNA for 409 cancer related genes. ( B ) MSI status judged by on-chip electrophoresis of the Bethesda panel markers. ( C ) Mutations in the mononucleotide repeats in coding regions of TGFBR2 and BAX determined using spheroid and FFPE tumor DNA. ( D ) IHC results of the cultured spheroids of tumor-initiating cells and FFPE primary tumors. Color keys are shown in boxes. See text for details.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, Chromatin Immunoprecipitation, Electrophoresis, Immunohistochemistry, Cell Culture

    61) Product Images from "Circulating tumour DNA reflects treatment response and clonal evolution in chronic lymphocytic leukaemia"

    Article Title: Circulating tumour DNA reflects treatment response and clonal evolution in chronic lymphocytic leukaemia

    Journal: Nature Communications

    doi: 10.1038/ncomms14756

    ctDNA dynamics reflect changes in disease burden across different tissue compartments. ( a ) Case CLL020: plasma ctDNA levels by TP53 p.R209fs mutation (left) and IGH (right) decreased over the course of ofatumumab and venetoclax treatment, which paralleled the decline in mutant DNA levels in the MNL, the decrease in PBL count and the reduction in lymphadenopathy as assessed by imaging. ( b ) Case CLL018: the initial peripheral lymphocytosis observed post ibrutinib treatment coincided with a increase in the abundance of the NOTCH1 p.P2415fs mutation in the MNL. This contrasted with plasma ctDNA levels of both the NOTCH1 p.P2415fs mutation (left) and IGH (right), which instead reflected the reduction in radiological disease burden. ( c ) Case CLL001: following obinutuzumab therapy, rapid clearance of the circulating leukaemic cells was observed and the NOTCH1 p.P2415fs mutation became undetectable in the MNL. This coincided with a parallel decrease in plasma ctDNA as assessed by NOTCH1 p.P2415fs mutation (left) and IGH (right) levels, although ctDNA did not become undetectable. The patient then had compartmentalized disease progression with increasing lymphadenopathy that was matched by a plasma ctDNA rise. Subsequent ibrutinib treatment resulted in a reduction in lymphadenopathy and plasma ctDNA. These dynamic changes were not reflected in the PBL count or by the NOTCH1 p.P2415fs mutation in MNL DNA. ( d ) Correlation between the change in ctDNA/MNL MAF versus change in radiological disease burden (cm 2 ) from the maximal value analysed for each individual patient across any time point. Of 38 matched serial time points from a total of 12 patients, the correlation with radiology was significantly better with ctDNA ( r 2 =0.78, P
    Figure Legend Snippet: ctDNA dynamics reflect changes in disease burden across different tissue compartments. ( a ) Case CLL020: plasma ctDNA levels by TP53 p.R209fs mutation (left) and IGH (right) decreased over the course of ofatumumab and venetoclax treatment, which paralleled the decline in mutant DNA levels in the MNL, the decrease in PBL count and the reduction in lymphadenopathy as assessed by imaging. ( b ) Case CLL018: the initial peripheral lymphocytosis observed post ibrutinib treatment coincided with a increase in the abundance of the NOTCH1 p.P2415fs mutation in the MNL. This contrasted with plasma ctDNA levels of both the NOTCH1 p.P2415fs mutation (left) and IGH (right), which instead reflected the reduction in radiological disease burden. ( c ) Case CLL001: following obinutuzumab therapy, rapid clearance of the circulating leukaemic cells was observed and the NOTCH1 p.P2415fs mutation became undetectable in the MNL. This coincided with a parallel decrease in plasma ctDNA as assessed by NOTCH1 p.P2415fs mutation (left) and IGH (right) levels, although ctDNA did not become undetectable. The patient then had compartmentalized disease progression with increasing lymphadenopathy that was matched by a plasma ctDNA rise. Subsequent ibrutinib treatment resulted in a reduction in lymphadenopathy and plasma ctDNA. These dynamic changes were not reflected in the PBL count or by the NOTCH1 p.P2415fs mutation in MNL DNA. ( d ) Correlation between the change in ctDNA/MNL MAF versus change in radiological disease burden (cm 2 ) from the maximal value analysed for each individual patient across any time point. Of 38 matched serial time points from a total of 12 patients, the correlation with radiology was significantly better with ctDNA ( r 2 =0.78, P

    Techniques Used: Mutagenesis, Imaging

    62) Product Images from "A comprehensive look at transcription factor gene expression changes in colorectal adenomas"

    Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-46

    Immunohistochemical staining for DACH1 protein in normal and neoplastic colon. (A) In normal mucosa, DACH1 expression is present in the nuclei of proliferating cells in the lower portion of the epithelial crypts (black arrowhead) and completely absent in the differentiated cells in the upper crypts (red arrowhead). (B) High-level DACH1 expression is seen in rapidly proliferating cells of adenomatous glands taking over normal crypts. Abundant expression is also seen in most cells of a colorectal adenoma (C) and a colorectal carcinoma (D) . In another colorectal cancer (E) , DACH1 expression is absent in neoplastic glands, although proliferating cells in the normal mucosa and in the tumoral stroma are positive. (F) A third colorectal cancer with patchy staining for DACH1.
    Figure Legend Snippet: Immunohistochemical staining for DACH1 protein in normal and neoplastic colon. (A) In normal mucosa, DACH1 expression is present in the nuclei of proliferating cells in the lower portion of the epithelial crypts (black arrowhead) and completely absent in the differentiated cells in the upper crypts (red arrowhead). (B) High-level DACH1 expression is seen in rapidly proliferating cells of adenomatous glands taking over normal crypts. Abundant expression is also seen in most cells of a colorectal adenoma (C) and a colorectal carcinoma (D) . In another colorectal cancer (E) , DACH1 expression is absent in neoplastic glands, although proliferating cells in the normal mucosa and in the tumoral stroma are positive. (F) A third colorectal cancer with patchy staining for DACH1.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    Transcript levels in colorectal adenomas and normal mucosa for the five target (hub) genes in the TF network shown in Figure 7 . Scatter plots of normalized log 2 expression intensity values (y-axis) obtained by Affymetrix Exon 1.0 array analysis of 17 colorectal adenomas and their corresponding samples of normal mucosa. Means and standard errors are represented by horizontal lines and t-bars, respectively. Mean fold changes in adenomas (vs. normal mucosa) are shown for each gene.
    Figure Legend Snippet: Transcript levels in colorectal adenomas and normal mucosa for the five target (hub) genes in the TF network shown in Figure 7 . Scatter plots of normalized log 2 expression intensity values (y-axis) obtained by Affymetrix Exon 1.0 array analysis of 17 colorectal adenomas and their corresponding samples of normal mucosa. Means and standard errors are represented by horizontal lines and t-bars, respectively. Mean fold changes in adenomas (vs. normal mucosa) are shown for each gene.

    Techniques Used: Expressing

    Hierarchical clustering analysis of colorectal tissue samples based on the TF genes found in two of the three sets shown in Figure 1 . (Pearson correlation, Ward distance). The 34 tissue samples represented on the x -axis include 17 normal mucosal samples and 17 adenomas. Each transcript probe set plotted on the y -axis is color-coded to reflect expression levels of the TF genes relative to their median expression levels across the entire tissue-sample set (red: high; green: low). Two hundred fifty-two of the 261 TF genes listed in Additional file 8 : Table S8 are reported here: the other 9 (i.e., the last 9 in Additional file 8 : Table S8) were not among the 35,285 genes represented on the Affymetrix Exon 1.0 microarray platform, but they were considered in networks generated with the MetaCore TF analysis.
    Figure Legend Snippet: Hierarchical clustering analysis of colorectal tissue samples based on the TF genes found in two of the three sets shown in Figure 1 . (Pearson correlation, Ward distance). The 34 tissue samples represented on the x -axis include 17 normal mucosal samples and 17 adenomas. Each transcript probe set plotted on the y -axis is color-coded to reflect expression levels of the TF genes relative to their median expression levels across the entire tissue-sample set (red: high; green: low). Two hundred fifty-two of the 261 TF genes listed in Additional file 8 : Table S8 are reported here: the other 9 (i.e., the last 9 in Additional file 8 : Table S8) were not among the 35,285 genes represented on the Affymetrix Exon 1.0 microarray platform, but they were considered in networks generated with the MetaCore TF analysis.

    Techniques Used: Expressing, Microarray, Generated

    DACH1 mRNA expression in normal colorectal mucosa, colorectal adenomas, and mismatch repair (MMR)-deficient and -proficient colorectal cancers. Scatter plot of normalized log 2 expression intensity values for DACH1 (Affymetrix U133 Plus 2.0 array analysis) in the 4 tissue groups analyzed in our previous study [ 3 ]. Means and standard errors are represented by horizontal lines and t-bars, respectively.
    Figure Legend Snippet: DACH1 mRNA expression in normal colorectal mucosa, colorectal adenomas, and mismatch repair (MMR)-deficient and -proficient colorectal cancers. Scatter plot of normalized log 2 expression intensity values for DACH1 (Affymetrix U133 Plus 2.0 array analysis) in the 4 tissue groups analyzed in our previous study [ 3 ]. Means and standard errors are represented by horizontal lines and t-bars, respectively.

    Techniques Used: Expressing

    Methylation analysis of the CpG island in the DACH1 promoter. (A) : Schematic depiction of the CpG islands located respectively 5’ upstream from the DACH1 transcription start site (CpG I) and in the first intron of the DACH1 gene (CpG II). (B) : Examples of CpG I COBRA analysis in colorectal cancers with intense (red), patchy (green), or no (blue) DACH1 protein immunostaining and in 4 colon cancer cell lines characterized by low (HT29 and Caco2) or very low (HCT116 and CO115) DACH1 expression (based on microarray-documented DACH1 mRNA expression levels - see also Additional file 9 ). Asterisks indicate Taq α I -digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. MW, molecular weight; bp, base pair.
    Figure Legend Snippet: Methylation analysis of the CpG island in the DACH1 promoter. (A) : Schematic depiction of the CpG islands located respectively 5’ upstream from the DACH1 transcription start site (CpG I) and in the first intron of the DACH1 gene (CpG II). (B) : Examples of CpG I COBRA analysis in colorectal cancers with intense (red), patchy (green), or no (blue) DACH1 protein immunostaining and in 4 colon cancer cell lines characterized by low (HT29 and Caco2) or very low (HCT116 and CO115) DACH1 expression (based on microarray-documented DACH1 mRNA expression levels - see also Additional file 9 ). Asterisks indicate Taq α I -digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. MW, molecular weight; bp, base pair.

    Techniques Used: Methylation, Combined Bisulfite Restriction Analysis Assay, Immunostaining, Expressing, Microarray, Molecular Weight

    Three-pronged procedure used to select 261 transcription factor (TF) genes with probable but relatively unexplored roles in colorectal tumorigenesis. The initial data set included 35,285 genes (including 23,768 annotated protein-encoding genes) represented on the Affymetrix Exon 1.0 microarray used to analyze 17 colorectal adenomas and corresponding specimens of normal mucosa. Left prong: Selection of 315 genes that encode TFs, are expressed in normal and/or adenomatous colorectal mucosa, and display significantly up- or downregulated transcription in adenomas. Middle and right prongs: MetaCore TF analysis identified 793 TF genes whose interaction networks were enriched for genes that were significantly up- or downregulated in adenomas. This list was then filtered to identify those with z scores of ≥2 (n = 257) and those with NormPDIs of > 0 (n = 495) (see Methods section for details).
    Figure Legend Snippet: Three-pronged procedure used to select 261 transcription factor (TF) genes with probable but relatively unexplored roles in colorectal tumorigenesis. The initial data set included 35,285 genes (including 23,768 annotated protein-encoding genes) represented on the Affymetrix Exon 1.0 microarray used to analyze 17 colorectal adenomas and corresponding specimens of normal mucosa. Left prong: Selection of 315 genes that encode TFs, are expressed in normal and/or adenomatous colorectal mucosa, and display significantly up- or downregulated transcription in adenomas. Middle and right prongs: MetaCore TF analysis identified 793 TF genes whose interaction networks were enriched for genes that were significantly up- or downregulated in adenomas. This list was then filtered to identify those with z scores of ≥2 (n = 257) and those with NormPDIs of > 0 (n = 495) (see Methods section for details).

    Techniques Used: Microarray, Selection

    63) Product Images from "Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent"

    Article Title: Epigenetic changes around the pX region and spontaneous HTLV-1 transcription are CTCF-independent

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.14741.2

    DNA methylation across the body of the HTLV-1 provirus. ( a ) Upper panel: count of CpG dinucleotides in a window of 350 bp in the HTLV-1 reference genome (L36905). Lower panel: schematic diagram of HTLV-1 provirus indicating the two LTRs and the 9 loci examined by MeDIP. ( b ) DNA methylation on the HTLV-1 provirus in the Tax + and Tax – populations from two HTLV-1-infected T cell clones (Clones TBX4B and 11.65). ( c ) DNA methylation on the HTLV-1 provirus in the CADM1 + Tax + and CADM1 + Tax – populations in PBMCs from two unrelated individuals (Patients TDZ and TED). The asterisk (*) indicates that the PCR failed to amplify.
    Figure Legend Snippet: DNA methylation across the body of the HTLV-1 provirus. ( a ) Upper panel: count of CpG dinucleotides in a window of 350 bp in the HTLV-1 reference genome (L36905). Lower panel: schematic diagram of HTLV-1 provirus indicating the two LTRs and the 9 loci examined by MeDIP. ( b ) DNA methylation on the HTLV-1 provirus in the Tax + and Tax – populations from two HTLV-1-infected T cell clones (Clones TBX4B and 11.65). ( c ) DNA methylation on the HTLV-1 provirus in the CADM1 + Tax + and CADM1 + Tax – populations in PBMCs from two unrelated individuals (Patients TDZ and TED). The asterisk (*) indicates that the PCR failed to amplify.

    Techniques Used: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Infection, Clone Assay, Polymerase Chain Reaction

    DNA methylation in the HTLV-1 LTR of patient-derived PBMCs. ( a ) The HTLV-1 LTR sequence (Accession no. L36905) with CpG dinucleotides highlighted in bold. The three TREs are coloured in red; the TATA box is indicated in the rectangle. ( b ) Schematic diagram of HTLV-1 provirus and the regions amplified with indicated sets of primers for bisulfite-sequencing. Sequencing results for each region are shown in the corresponding panels ( c – f ). The three TREs are indicated by red bars. ( c – f ) Schematic representation of DNA methylation for each clone sequenced. Open circles indicate unmethylated cytosine; closed circles methylated cytosine. The numbers indicate the corresponding CpG sites in panel ( a ).
    Figure Legend Snippet: DNA methylation in the HTLV-1 LTR of patient-derived PBMCs. ( a ) The HTLV-1 LTR sequence (Accession no. L36905) with CpG dinucleotides highlighted in bold. The three TREs are coloured in red; the TATA box is indicated in the rectangle. ( b ) Schematic diagram of HTLV-1 provirus and the regions amplified with indicated sets of primers for bisulfite-sequencing. Sequencing results for each region are shown in the corresponding panels ( c – f ). The three TREs are indicated by red bars. ( c – f ) Schematic representation of DNA methylation for each clone sequenced. Open circles indicate unmethylated cytosine; closed circles methylated cytosine. The numbers indicate the corresponding CpG sites in panel ( a ).

    Techniques Used: DNA Methylation Assay, Derivative Assay, Sequencing, Amplification, Methylation Sequencing, Methylation

    Overview of the cell preparations. ( a ) Preparation of Tax + and Tax – populations from PBMCs obtained from HTLV-1-infected patients. PBMCs were stained for CD4, CADM1 and Tax after overnight culture. Tax + and Tax – fractions were collected from the CADM1 + population. ( b ) Preparation of the Tax + and Tax – populations from HTLV-1-infected T cell clones. HTLV-1-infected T cell clones were stained for intracellular Tax and sorted according to Tax expression.
    Figure Legend Snippet: Overview of the cell preparations. ( a ) Preparation of Tax + and Tax – populations from PBMCs obtained from HTLV-1-infected patients. PBMCs were stained for CD4, CADM1 and Tax after overnight culture. Tax + and Tax – fractions were collected from the CADM1 + population. ( b ) Preparation of the Tax + and Tax – populations from HTLV-1-infected T cell clones. HTLV-1-infected T cell clones were stained for intracellular Tax and sorted according to Tax expression.

    Techniques Used: Infection, Staining, Clone Assay, Expressing

    HTLV-1 transcription in two distinct models. a ) Schematic diagram of HTLV-1 provirus inserted in the host genome. The HTLV-1 provirus has two identical LTRs, one at each end of the provirus. As well as genes encoding the canonical retroviral structural components Gag, Pol and Env, the provirus contains a group of regulatory genes in the pX region on the plus-strand. The plus-strand transcripts, represented by tax , are coloured in red, and the minus-strand transcript HBZ in yellow. ( b ) In PBMCs freshly isolated from HTLV-1 carriers, HTLV-1 reactivates and expresses the plus-strand transcripts within a few hours of culture; but these transcripts remain transcriptionally silent for most of the time in vivo . ( c ) In HTLV-1-infected T cell clones cultured in vitro , the promoter activity for plus-strand transcripts shuttles between the on and off state. The plus-strand transcripts are only produced when the promoter activity is on, yielding only a limited fraction of cells that are positive for the plus-strand transcripts at a given time.
    Figure Legend Snippet: HTLV-1 transcription in two distinct models. a ) Schematic diagram of HTLV-1 provirus inserted in the host genome. The HTLV-1 provirus has two identical LTRs, one at each end of the provirus. As well as genes encoding the canonical retroviral structural components Gag, Pol and Env, the provirus contains a group of regulatory genes in the pX region on the plus-strand. The plus-strand transcripts, represented by tax , are coloured in red, and the minus-strand transcript HBZ in yellow. ( b ) In PBMCs freshly isolated from HTLV-1 carriers, HTLV-1 reactivates and expresses the plus-strand transcripts within a few hours of culture; but these transcripts remain transcriptionally silent for most of the time in vivo . ( c ) In HTLV-1-infected T cell clones cultured in vitro , the promoter activity for plus-strand transcripts shuttles between the on and off state. The plus-strand transcripts are only produced when the promoter activity is on, yielding only a limited fraction of cells that are positive for the plus-strand transcripts at a given time.

    Techniques Used: Isolation, In Vivo, Infection, Clone Assay, Cell Culture, In Vitro, Activity Assay, Produced

    Kinetics of the plus- and minus-strand transcription in HTLV-1-infected T cell clones. ( a ) Representative images of HTLV-1 transcripts by single-molecule RNA-FISH (maximum-projection of Z-stacks). Red spots indicate the plus-strand transcripts, and yellow spots the minus-strand transcripts. Blue indicates the DAPI-stained nucleus. Plus- and minus-signs in brackets indicate respectively the presence or absence of the mRNA. Scale bar (white) = 5 µm. ( b ) Spot counts of the plus-strand (upper row) and the minus-strand transcripts (lower row) respectively in the unaltered and ΔCTCF-binding subclones. The insets in the upper row capture low-frequency events on a magnified y-axis. The bar in the first bin in the insets is greyed out because it is out of scale.
    Figure Legend Snippet: Kinetics of the plus- and minus-strand transcription in HTLV-1-infected T cell clones. ( a ) Representative images of HTLV-1 transcripts by single-molecule RNA-FISH (maximum-projection of Z-stacks). Red spots indicate the plus-strand transcripts, and yellow spots the minus-strand transcripts. Blue indicates the DAPI-stained nucleus. Plus- and minus-signs in brackets indicate respectively the presence or absence of the mRNA. Scale bar (white) = 5 µm. ( b ) Spot counts of the plus-strand (upper row) and the minus-strand transcripts (lower row) respectively in the unaltered and ΔCTCF-binding subclones. The insets in the upper row capture low-frequency events on a magnified y-axis. The bar in the first bin in the insets is greyed out because it is out of scale.

    Techniques Used: Infection, Clone Assay, Fluorescence In Situ Hybridization, Staining, Binding Assay

    Histone modifications and CTCF-binding in the HTLV-1 provirus. Chromatin immunoprecipitation (ChIP) was used to identify ( a ) histone modifications and CTCF-binding in the Tax + and Tax – populations from an HTLV-1-infected T cell clone (TBX4B); and ( b ) histone modifications in the CADM1 + Tax + and CADM1 + Tax – populations from PBMCs obtained from HTLV-1-infected patients (Patients TW and TCD). The horizontal axis indicates the nucleotide position in the full-length HTLV-1 provirus (J02029), and the vertical axis the read depth (arbitrary units). The reads that aligned within either one of the LTRs are greyed out. The black bars on the horizontal axis indicates the LTRs.
    Figure Legend Snippet: Histone modifications and CTCF-binding in the HTLV-1 provirus. Chromatin immunoprecipitation (ChIP) was used to identify ( a ) histone modifications and CTCF-binding in the Tax + and Tax – populations from an HTLV-1-infected T cell clone (TBX4B); and ( b ) histone modifications in the CADM1 + Tax + and CADM1 + Tax – populations from PBMCs obtained from HTLV-1-infected patients (Patients TW and TCD). The horizontal axis indicates the nucleotide position in the full-length HTLV-1 provirus (J02029), and the vertical axis the read depth (arbitrary units). The reads that aligned within either one of the LTRs are greyed out. The black bars on the horizontal axis indicates the LTRs.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Infection

    CTCF occupancy in the HTLV-1 provirus in patient-derived PBMCs. CTCF occupancy was examined by droplet digital PCR following ChIP for CTCF. The experiment was carried out on PBMCs after overnight incubation in vitro . Replicate 1 is obtained from pooled samples of 4 patients (TCR, TEJ, TED and TW) and Replicate 2 from 3 patients (TED, TCR and TEJ).
    Figure Legend Snippet: CTCF occupancy in the HTLV-1 provirus in patient-derived PBMCs. CTCF occupancy was examined by droplet digital PCR following ChIP for CTCF. The experiment was carried out on PBMCs after overnight incubation in vitro . Replicate 1 is obtained from pooled samples of 4 patients (TCR, TEJ, TED and TW) and Replicate 2 from 3 patients (TED, TCR and TEJ).

    Techniques Used: Derivative Assay, Digital PCR, Chromatin Immunoprecipitation, Incubation, In Vitro

    Alteration of the CTCF-binding site in the provirus in HTLV-1-infected T cell clones. ( a ) The sequence of the CTCF-binding site in the HTLV-1 provirus. The upper panel is from a subclone with the sequence unchanged, and the lower panel from a subclone in which the sequence was altered by CRISPR/Cas9 modification. ( b ) Flow cytometric analysis of the mutated clone after staining for intracellular Tax protein.
    Figure Legend Snippet: Alteration of the CTCF-binding site in the provirus in HTLV-1-infected T cell clones. ( a ) The sequence of the CTCF-binding site in the HTLV-1 provirus. The upper panel is from a subclone with the sequence unchanged, and the lower panel from a subclone in which the sequence was altered by CRISPR/Cas9 modification. ( b ) Flow cytometric analysis of the mutated clone after staining for intracellular Tax protein.

    Techniques Used: Binding Assay, Infection, Clone Assay, Sequencing, CRISPR, Modification, Flow Cytometry, Staining

    64) Product Images from "Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens"

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034683

    MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.
    Figure Legend Snippet: Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Cell Culture, RNA Extraction, Isolation, Agarose Gel Electrophoresis

    DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.
    Figure Legend Snippet: DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Extraction, Isolation

    Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.
    Figure Legend Snippet: Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Techniques Used: Methylation, Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Isolation, DNA Methylation Assay

    Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.
    Figure Legend Snippet: Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Electrophoresis

    Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: RNA Expression, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    65) Product Images from "DNA Extraction Columns Contaminated with Murine Sequences"

    Article Title: DNA Extraction Columns Contaminated with Murine Sequences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023484

    Amplification of contaminating DNA from empty columns of the QiaAmp FFPE Tissue Kit. Lanes 1–10, naïve DNA extraction columns; lanes 11–14, PCR water controls; lane 15, positive control; upper panel, McCoy cellular DNA; middle and lower panel, XMRV VP62 infectious clone; lane 16, 100 bp DNA ladder (Invitrogen, Paisley, UK). Upper panel, detection of contaminating sequences using IAP-specific primers IAP for and IAP rev. All columns apart from column no 7 produce amplicons. Size differences reflect the fact that IAP sequences form a class of slightly different retrotransposons. Middle panel, PCR products using primers XTP1 and MLV reverse outer under relaxed annealing conditions. Lower panel, multiplex PCR using the four primers XMRV-R, XMRV Forward outer, XMRV Reverse outer and 1154R under less stringent annealing conditions.
    Figure Legend Snippet: Amplification of contaminating DNA from empty columns of the QiaAmp FFPE Tissue Kit. Lanes 1–10, naïve DNA extraction columns; lanes 11–14, PCR water controls; lane 15, positive control; upper panel, McCoy cellular DNA; middle and lower panel, XMRV VP62 infectious clone; lane 16, 100 bp DNA ladder (Invitrogen, Paisley, UK). Upper panel, detection of contaminating sequences using IAP-specific primers IAP for and IAP rev. All columns apart from column no 7 produce amplicons. Size differences reflect the fact that IAP sequences form a class of slightly different retrotransposons. Middle panel, PCR products using primers XTP1 and MLV reverse outer under relaxed annealing conditions. Lower panel, multiplex PCR using the four primers XMRV-R, XMRV Forward outer, XMRV Reverse outer and 1154R under less stringent annealing conditions.

    Techniques Used: Amplification, Formalin-fixed Paraffin-Embedded, DNA Extraction, Polymerase Chain Reaction, Positive Control, Multiplex Assay

    66) Product Images from "Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations"

    Article Title: Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159094

    Multiplex assays for the most frequent EGFR mutations. In panel A, B and C, 2D-plots of the three-plex for follow up of the three sensitivity mutations with the T790M resistance mutation. The four-plex is shown in panel D. A pool of fragmented DNA extracted from two cell lines (H1975 harboring L858R and T790M mutations, H1650 harboring Del19 mutation), DNA from FFPE sample (for L861Q mutation) and fragmented wild-type only genomic DNA was used as input. A mix of mutation-specific VIC and/or 6-carboxyfluorescein cast™ and ZEN™ probes was optimized. In the table, event counts from the single experiments are listed (input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer). A . U , arbitrary units; WT , wild-type; S , sensitivity mutation; R , resistance mutation .
    Figure Legend Snippet: Multiplex assays for the most frequent EGFR mutations. In panel A, B and C, 2D-plots of the three-plex for follow up of the three sensitivity mutations with the T790M resistance mutation. The four-plex is shown in panel D. A pool of fragmented DNA extracted from two cell lines (H1975 harboring L858R and T790M mutations, H1650 harboring Del19 mutation), DNA from FFPE sample (for L861Q mutation) and fragmented wild-type only genomic DNA was used as input. A mix of mutation-specific VIC and/or 6-carboxyfluorescein cast™ and ZEN™ probes was optimized. In the table, event counts from the single experiments are listed (input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer). A . U , arbitrary units; WT , wild-type; S , sensitivity mutation; R , resistance mutation .

    Techniques Used: Multiplex Assay, Mutagenesis, Formalin-fixed Paraffin-Embedded, Digital PCR

    Multiplex panel for the most common EGFR mutations using HdX™ Reference Standards. A three-plex panel for follow-up of the 3 EGFR sensitivity mutations with the T790M resistance mutation. As input, DNA from FFPE Reference Standards (R.S.) (panels A, B, C), Multiplex I cfDNA (panels D and F) and Multiplex genomic DNA (panel E) from Horizon Diagnostics. The FFPE R.S. contains 50% of genomic DNA and 5% of each mutation, while while the Multiplex DNA is engineered from mutant cell lines for generation of 12.5% EGFR allelic frequency for the four mutations. Multiplex I cfDNA provides a set containing fragmented DNA in a range of low allelic frequencies (from 0.1% to 5%): we showed here only the 5% allelic frequency DNA. In the table, event counts from the single experiments are listed (input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer). A . U , arbitrary units; WT , wild-type; gDNA , genomic DNA .
    Figure Legend Snippet: Multiplex panel for the most common EGFR mutations using HdX™ Reference Standards. A three-plex panel for follow-up of the 3 EGFR sensitivity mutations with the T790M resistance mutation. As input, DNA from FFPE Reference Standards (R.S.) (panels A, B, C), Multiplex I cfDNA (panels D and F) and Multiplex genomic DNA (panel E) from Horizon Diagnostics. The FFPE R.S. contains 50% of genomic DNA and 5% of each mutation, while while the Multiplex DNA is engineered from mutant cell lines for generation of 12.5% EGFR allelic frequency for the four mutations. Multiplex I cfDNA provides a set containing fragmented DNA in a range of low allelic frequencies (from 0.1% to 5%): we showed here only the 5% allelic frequency DNA. In the table, event counts from the single experiments are listed (input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer). A . U , arbitrary units; WT , wild-type; gDNA , genomic DNA .

    Techniques Used: Multiplex Assay, Mutagenesis, Formalin-fixed Paraffin-Embedded, Digital PCR

    Examples of titration series with EGFR castPCR™, ZEN™ and TaqMan® probes. Serial dilutions of L858R, T790M, Del19, or L861Q mutated DNA (extracted from H1975 cell line, H1650 cell line or FFPE tissue, respectively) in human wild-type genomic DNA. Individual data points are displayed for independent replicates. The expected mutant to wild-type ratio (black line) is shown. Green continuous and dashed lines represent LOB and LOD values, respectively, evaluated from droplets falling into the mutated-DNA cluster and analyzed in a WT gDNA sample for each replicate. For the lowest titration point (0.01%), we used a higher amount of input DNA. Thus, corresponding LOB and LOD values are represented by red lines. Since number of FP was increasing with quantity of input DNA for EGFR p.T790M castPCR™ test, LOB and LOD calculation could not be performed (refer to [ 8 ]).
    Figure Legend Snippet: Examples of titration series with EGFR castPCR™, ZEN™ and TaqMan® probes. Serial dilutions of L858R, T790M, Del19, or L861Q mutated DNA (extracted from H1975 cell line, H1650 cell line or FFPE tissue, respectively) in human wild-type genomic DNA. Individual data points are displayed for independent replicates. The expected mutant to wild-type ratio (black line) is shown. Green continuous and dashed lines represent LOB and LOD values, respectively, evaluated from droplets falling into the mutated-DNA cluster and analyzed in a WT gDNA sample for each replicate. For the lowest titration point (0.01%), we used a higher amount of input DNA. Thus, corresponding LOB and LOD values are represented by red lines. Since number of FP was increasing with quantity of input DNA for EGFR p.T790M castPCR™ test, LOB and LOD calculation could not be performed (refer to [ 8 ]).

    Techniques Used: Titration, Formalin-fixed Paraffin-Embedded, Mutagenesis

    67) Product Images from "Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR"

    Article Title: Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163070

    KRAS multiplex mutation analysis with colorectal carcinoma FFPE. Genomic DNA from FFPE tissues were used for AsP and Mult-AsP PCR assay. In all cases the qPCR assays contained the non-mutated reference control reaction (red line). Each DNA was amplified in AsP and Multi-AsP PCR (green and blue lines, respectively). In gray curves indicated NTC reaction.
    Figure Legend Snippet: KRAS multiplex mutation analysis with colorectal carcinoma FFPE. Genomic DNA from FFPE tissues were used for AsP and Mult-AsP PCR assay. In all cases the qPCR assays contained the non-mutated reference control reaction (red line). Each DNA was amplified in AsP and Multi-AsP PCR (green and blue lines, respectively). In gray curves indicated NTC reaction.

    Techniques Used: Multiplex Assay, Mutagenesis, Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification

    68) Product Images from "Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing"

    Article Title: Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121891

    Read number and error rate of the NGS assay. (A) Average read number of 9 amplicons in 10 normal DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon. (B) Error rate of position detection in 10 normal DNA samples (average with standard deviation). Horizontal axis, base position; vertical axis, error rate. (C) Average read number of 9 amplicons in 20 colorectal cancer FFPE DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon.
    Figure Legend Snippet: Read number and error rate of the NGS assay. (A) Average read number of 9 amplicons in 10 normal DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon. (B) Error rate of position detection in 10 normal DNA samples (average with standard deviation). Horizontal axis, base position; vertical axis, error rate. (C) Average read number of 9 amplicons in 20 colorectal cancer FFPE DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon.

    Techniques Used: Next-Generation Sequencing, Standard Deviation, Polymerase Chain Reaction, Amplification, Formalin-fixed Paraffin-Embedded

    69) Product Images from "Next-Generation Sequencing of RNA and DNA Isolated from Paired Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Samples of Human Cancer and Normal Tissue"

    Article Title: Next-Generation Sequencing of RNA and DNA Isolated from Paired Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Samples of Human Cancer and Normal Tissue

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098187

    Single nucleotide variants detected in DNA-Exome-Seq data from the paired FF/FFPE samples. The percentage of common (grey), exclusively FF (white) and exclusively FFPE (red) single nucleotide variants are shown as bars referring to the left axis. The average coverage is shown as stars for FF (white) and FFPE (red) on the right axis. Patient ID and number of years of storage are shown below.
    Figure Legend Snippet: Single nucleotide variants detected in DNA-Exome-Seq data from the paired FF/FFPE samples. The percentage of common (grey), exclusively FF (white) and exclusively FFPE (red) single nucleotide variants are shown as bars referring to the left axis. The average coverage is shown as stars for FF (white) and FFPE (red) on the right axis. Patient ID and number of years of storage are shown below.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    Effects of storage time on post-mapping results from the paired FF/FFPE samples. Fractions of non-specifically mapped DNA-Exome-Seq (A) and RNA-Seq (B) reads; fractions of non-perfectly mapped DNA-Exome-Seq (C) and RNA-Seq (D) reads for FF (black) and FFPE (red) for each of the ten samples by number of years since sampling.
    Figure Legend Snippet: Effects of storage time on post-mapping results from the paired FF/FFPE samples. Fractions of non-specifically mapped DNA-Exome-Seq (A) and RNA-Seq (B) reads; fractions of non-perfectly mapped DNA-Exome-Seq (C) and RNA-Seq (D) reads for FF (black) and FFPE (red) for each of the ten samples by number of years since sampling.

    Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Sequencing Assay, Sampling

    70) Product Images from "Targeted next-generation sequencing in cytology specimens for molecular profiling of lung adenocarcinoma"

    Article Title: Targeted next-generation sequencing in cytology specimens for molecular profiling of lung adenocarcinoma

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Clinical observation of DNA quality index (DQI) in 211 consecutive surplus samples. The DNA quality of 211 consecutive surplus specimens-including 130 FFPE tissue blocks (FFPE Tissue), 27 FFPE cell blocks of FNA samples (FFPE FNA), 29 freshly centrifuged pleural fluid samples (fresh pleural fluid), and 25 FFPE cell blocks of pleural fluid (FFPE pleural fluid)-was evaluated. The DNA Quality Index and amplifiable DNA concentrations were determined using a DNA Quality Index Kit (Beijing ACCB Biotech Ltd., Beijing, China) according to the manufacturer’s instructions.
    Figure Legend Snippet: Clinical observation of DNA quality index (DQI) in 211 consecutive surplus samples. The DNA quality of 211 consecutive surplus specimens-including 130 FFPE tissue blocks (FFPE Tissue), 27 FFPE cell blocks of FNA samples (FFPE FNA), 29 freshly centrifuged pleural fluid samples (fresh pleural fluid), and 25 FFPE cell blocks of pleural fluid (FFPE pleural fluid)-was evaluated. The DNA Quality Index and amplifiable DNA concentrations were determined using a DNA Quality Index Kit (Beijing ACCB Biotech Ltd., Beijing, China) according to the manufacturer’s instructions.

    Techniques Used: Formalin-fixed Paraffin-Embedded

    71) Product Images from "A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue"

    Article Title: A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    Journal: Analytical Biochemistry

    doi: 10.1016/j.ab.2012.03.012

    Schematic diagram of DNA extraction procedures from FFPE tissue section or core.
    Figure Legend Snippet: Schematic diagram of DNA extraction procedures from FFPE tissue section or core.

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Analysis of the quality of the DNA extracted from archival human colon FFPE tissues by the Nanodrop spectrophotometer. The DNA quality was accessed by distribution of the ratio values (A 260 /A 280 and A 260 /A 230 ).
    Figure Legend Snippet: Analysis of the quality of the DNA extracted from archival human colon FFPE tissues by the Nanodrop spectrophotometer. The DNA quality was accessed by distribution of the ratio values (A 260 /A 280 and A 260 /A 230 ).

    Techniques Used: Formalin-fixed Paraffin-Embedded, Spectrophotometry

    Comparison of DNA extraction yield and quality between a new rapid method and the QIAamp DNA FFPE tissue kit. We performed DNA extraction from archival human liver cores or sections. Representative data were presented as a bar graph ( A ) and a gel image
    Figure Legend Snippet: Comparison of DNA extraction yield and quality between a new rapid method and the QIAamp DNA FFPE tissue kit. We performed DNA extraction from archival human liver cores or sections. Representative data were presented as a bar graph ( A ) and a gel image

    Techniques Used: DNA Extraction, Formalin-fixed Paraffin-Embedded

    MSP analysis of sFRP1 and TFPI2 genes in colon samples using the rapid and classic DNA extraction. Methylation pattern of (A) sFRP1 and (B) TFPI2 gene promoter region in 18 FFPE colon samples using rapid DNA extraction method. Colon FFPE tumor samples
    Figure Legend Snippet: MSP analysis of sFRP1 and TFPI2 genes in colon samples using the rapid and classic DNA extraction. Methylation pattern of (A) sFRP1 and (B) TFPI2 gene promoter region in 18 FFPE colon samples using rapid DNA extraction method. Colon FFPE tumor samples

    Techniques Used: DNA Extraction, Methylation, Formalin-fixed Paraffin-Embedded

    72) Product Images from "Diagnostic value and lymph node metastasis prediction of a custom-made panel (thyroline) in thyroid cancer"

    Article Title: Diagnostic value and lymph node metastasis prediction of a custom-made panel (thyroline) in thyroid cancer

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6493

    Gene mutations and fusions in subtypes of TC and workflow of NGS. (A) BRAF, RAS, TERT, ETV6, EIF1AX, GNAS, PIK3CA, TP53 and NTRK1 mutations, as well as RET and ALK fusions, were found in PTC. BRAF, TERT, ALK fusion, GNAS, AKT1, PIK3CA, TP53 and PTEN were found in ATC. RAS, TERT, TSHR, GNAS, PENT and TP53 were found in FTC, while only RET and RAS mutations were found in MTC. (B) FFPE samples were obtained from 98 thyroid nodule patients, which was followed by CTC enumeration on NanoVelcro Chips. After collecting clinical information, we analyzed the correlation between pathological information and NGS results. (C) DNA from FFPE tissue was amplified for enrichment of target regions in a multiplex PCR reaction. Then, the library was prepared by ligating the PCR amplicons into platform-specific adapters and adding bar codes for specimen multiplexing. Finally, the library was enriched by clonal amplification (emPCR) and sequenced by massively parallel sequencing on the Ion Torrent PGM. The data analysis and variant calling were performed using bioinformatic pipelines followed by a custom SeqReporter algorithm for filtering and annotation of genetic variants. TC, thyroid cancer; NGS, next-generation sequencing; PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; FFPE, formalin-fixed, paraffin-embedded.
    Figure Legend Snippet: Gene mutations and fusions in subtypes of TC and workflow of NGS. (A) BRAF, RAS, TERT, ETV6, EIF1AX, GNAS, PIK3CA, TP53 and NTRK1 mutations, as well as RET and ALK fusions, were found in PTC. BRAF, TERT, ALK fusion, GNAS, AKT1, PIK3CA, TP53 and PTEN were found in ATC. RAS, TERT, TSHR, GNAS, PENT and TP53 were found in FTC, while only RET and RAS mutations were found in MTC. (B) FFPE samples were obtained from 98 thyroid nodule patients, which was followed by CTC enumeration on NanoVelcro Chips. After collecting clinical information, we analyzed the correlation between pathological information and NGS results. (C) DNA from FFPE tissue was amplified for enrichment of target regions in a multiplex PCR reaction. Then, the library was prepared by ligating the PCR amplicons into platform-specific adapters and adding bar codes for specimen multiplexing. Finally, the library was enriched by clonal amplification (emPCR) and sequenced by massively parallel sequencing on the Ion Torrent PGM. The data analysis and variant calling were performed using bioinformatic pipelines followed by a custom SeqReporter algorithm for filtering and annotation of genetic variants. TC, thyroid cancer; NGS, next-generation sequencing; PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; FFPE, formalin-fixed, paraffin-embedded.

    Techniques Used: Next-Generation Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Multiplex Assay, Polymerase Chain Reaction, Multiplexing, Sequencing, Variant Assay

    73) Product Images from "Using ddPCR to assess the DNA yield of FFPE samples"

    Article Title: Using ddPCR to assess the DNA yield of FFPE samples

    Journal: Biomolecular Detection and Quantification

    doi: 10.1016/j.bdq.2018.10.001

    Schematic workflow for testing various factors in extracting DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. a Commercially available from QIAGEN; b Included in the QIAamp DNA FFPE Tissue Kit. Abbreviations: PK: Proteinase K; PCE: Phenol-chloroform extraction and ethanol precipitation.
    Figure Legend Snippet: Schematic workflow for testing various factors in extracting DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. a Commercially available from QIAGEN; b Included in the QIAamp DNA FFPE Tissue Kit. Abbreviations: PK: Proteinase K; PCE: Phenol-chloroform extraction and ethanol precipitation.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Ethanol Precipitation

    74) Product Images from "Non-invasive Molecular Detection of Minimal Residual Disease in Papillary Thyroid Cancer Patients"

    Article Title: Non-invasive Molecular Detection of Minimal Residual Disease in Papillary Thyroid Cancer Patients

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2019.01510

    Percent plasma ctDNA predicts disease status. Representative FACS plots for selected patients showing the percent (%) plasma cell-free BRAFV600 wild type (cfDNA) and mutant DNA (ctDNA) levels for thyroid cancer patients whose tumors harbored BRAF V600E hotspot mutation with various levels of disease; (A) plasma ctDNA (0%) level for a patient with no evidence of disease (NED), (B) plasma ctDNA level (0.13%) for a patient with PD and no evidence of distant metastasis, (C) plasma ctDNA level (1.46%) for a patient with lung metastasis, and (D) plasma ctDNA level (2.07%) for another patient who also had lung metastasis. (E) Dot plot showing percent ctDNA levels for all 38 patients with either PD or NED.
    Figure Legend Snippet: Percent plasma ctDNA predicts disease status. Representative FACS plots for selected patients showing the percent (%) plasma cell-free BRAFV600 wild type (cfDNA) and mutant DNA (ctDNA) levels for thyroid cancer patients whose tumors harbored BRAF V600E hotspot mutation with various levels of disease; (A) plasma ctDNA (0%) level for a patient with no evidence of disease (NED), (B) plasma ctDNA level (0.13%) for a patient with PD and no evidence of distant metastasis, (C) plasma ctDNA level (1.46%) for a patient with lung metastasis, and (D) plasma ctDNA level (2.07%) for another patient who also had lung metastasis. (E) Dot plot showing percent ctDNA levels for all 38 patients with either PD or NED.

    Techniques Used: FACS, Mutagenesis

    Plasma cfDNA levels are higher in NED patients. A box-plot overlaid by a dot-plot showing the range and median values of plasma total cell-free DNA (cfDNA) (copies/ml) levels for all the patients ( n = 38) that were estimated by the 3D digital PCR. Plasma total cfDNA levels are significantly higher (Wilcoxon test, p = 0.041, n = 38) in patients with no evidence of disease (NED) compared to those who had persistent disease (PD). * p
    Figure Legend Snippet: Plasma cfDNA levels are higher in NED patients. A box-plot overlaid by a dot-plot showing the range and median values of plasma total cell-free DNA (cfDNA) (copies/ml) levels for all the patients ( n = 38) that were estimated by the 3D digital PCR. Plasma total cfDNA levels are significantly higher (Wilcoxon test, p = 0.041, n = 38) in patients with no evidence of disease (NED) compared to those who had persistent disease (PD). * p

    Techniques Used: Digital PCR

    75) Product Images from "A comprehensive look at transcription factor gene expression changes in colorectal adenomas"

    Article Title: A comprehensive look at transcription factor gene expression changes in colorectal adenomas

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-46

    Immunohistochemical staining for DACH1 protein in normal and neoplastic colon. (A) In normal mucosa, DACH1 expression is present in the nuclei of proliferating cells in the lower portion of the epithelial crypts (black arrowhead) and completely absent in the differentiated cells in the upper crypts (red arrowhead). (B) High-level DACH1 expression is seen in rapidly proliferating cells of adenomatous glands taking over normal crypts. Abundant expression is also seen in most cells of a colorectal adenoma (C) and a colorectal carcinoma (D) . In another colorectal cancer (E) , DACH1 expression is absent in neoplastic glands, although proliferating cells in the normal mucosa and in the tumoral stroma are positive. (F) A third colorectal cancer with patchy staining for DACH1.
    Figure Legend Snippet: Immunohistochemical staining for DACH1 protein in normal and neoplastic colon. (A) In normal mucosa, DACH1 expression is present in the nuclei of proliferating cells in the lower portion of the epithelial crypts (black arrowhead) and completely absent in the differentiated cells in the upper crypts (red arrowhead). (B) High-level DACH1 expression is seen in rapidly proliferating cells of adenomatous glands taking over normal crypts. Abundant expression is also seen in most cells of a colorectal adenoma (C) and a colorectal carcinoma (D) . In another colorectal cancer (E) , DACH1 expression is absent in neoplastic glands, although proliferating cells in the normal mucosa and in the tumoral stroma are positive. (F) A third colorectal cancer with patchy staining for DACH1.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    DACH1 mRNA expression in normal colorectal mucosa, colorectal adenomas, and mismatch repair (MMR)-deficient and -proficient colorectal cancers. Scatter plot of normalized log 2 expression intensity values for DACH1 (Affymetrix U133 Plus 2.0 array analysis) in the 4 tissue groups analyzed in our previous study [ 3 ]. Means and standard errors are represented by horizontal lines and t-bars, respectively.
    Figure Legend Snippet: DACH1 mRNA expression in normal colorectal mucosa, colorectal adenomas, and mismatch repair (MMR)-deficient and -proficient colorectal cancers. Scatter plot of normalized log 2 expression intensity values for DACH1 (Affymetrix U133 Plus 2.0 array analysis) in the 4 tissue groups analyzed in our previous study [ 3 ]. Means and standard errors are represented by horizontal lines and t-bars, respectively.

    Techniques Used: Expressing

    Methylation analysis of the CpG island in the DACH1 promoter. (A) : Schematic depiction of the CpG islands located respectively 5’ upstream from the DACH1 transcription start site (CpG I) and in the first intron of the DACH1 gene (CpG II). (B) : Examples of CpG I COBRA analysis in colorectal cancers with intense (red), patchy (green), or no (blue) DACH1 protein immunostaining and in 4 colon cancer cell lines characterized by low (HT29 and Caco2) or very low (HCT116 and CO115) DACH1 expression (based on microarray-documented DACH1 mRNA expression levels - see also Additional file 9 ). Asterisks indicate Taq α I -digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. MW, molecular weight; bp, base pair.
    Figure Legend Snippet: Methylation analysis of the CpG island in the DACH1 promoter. (A) : Schematic depiction of the CpG islands located respectively 5’ upstream from the DACH1 transcription start site (CpG I) and in the first intron of the DACH1 gene (CpG II). (B) : Examples of CpG I COBRA analysis in colorectal cancers with intense (red), patchy (green), or no (blue) DACH1 protein immunostaining and in 4 colon cancer cell lines characterized by low (HT29 and Caco2) or very low (HCT116 and CO115) DACH1 expression (based on microarray-documented DACH1 mRNA expression levels - see also Additional file 9 ). Asterisks indicate Taq α I -digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. MW, molecular weight; bp, base pair.

    Techniques Used: Methylation, Combined Bisulfite Restriction Analysis Assay, Immunostaining, Expressing, Microarray, Molecular Weight

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    Generated:

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    Nucleic Acid Electrophoresis:

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    Fluorescence:

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    Mutagenesis:

    Article Title: A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry
    Article Snippet: The DNA extraction was performed with QIAamp™ DNA FFPE Tissue kit and automated on the QIAcube robot (QIAGEN, Valencia, CA, USA), as previously described and according to the manufacturer’s instructions . .. Firstly, as part of standard clinical practice, we performed the mutation analysis by direct sequencing (the current gold standard).

    Isolation:

    Article Title: Critical Issues in Mycobiota Analysis
    Article Snippet: About 70 commercially kits are available for DNA extraction out of FFPE material (Kocjan et al., ), however, nucleic acid isolation from FFPE material is challenging. .. To highlight the influence of pre-analytics on ITS based mycobiota investigations we assessed the performance of DNA extraction from human skin FFPE samples (see Supplementary Table for sample information) with a commercially available kit (QIAamp DNA FFPE tissue kit, Qiagen) reported to be efficient for fungal DNA extraction out of FFPE material (Muñoz-Cadavid et al., ).

    Article Title: Analytic validation and real-time clinical application of an amplicon-based targeted gene panel for advanced cancer
    Article Snippet: .. DNA extraction and QC DNA was isolated from cell lines and frozen tumors using QiaAMP DNA mini kit (Qiagen, Hilden, Germany), from blood using QiaAMP blood mini kit (Qiagen), and from FFPE tumors using QiaAMP DNA FFPE Tissue kit (Qiagen) per manufacturer's instructions. .. A Qubit fluorometer (ThermoFisher, Waltham, MA) was used to quantitate DNA.

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies
    Article Snippet: .. Assessment of nucleic acids extraction kits from FFPE biopsies with normal histology The study material, consisting of 30 FFPE prostate biopsies with normal histology, was randomly divided into three different groups; 1) ten biopsies for comparison between the High Pure FFPE RNA Micro Kit (Roche Diagnostics, West Sussex, UK) and the RNeasy® FFPE kit (Qiagen, Hilden, Germany), 2) ten biopsies for comparison between the High Pure DNA FFPET Isolation Kit (Roche Diagnostics, West Sussex, UK) and the QIAamp® DNA FFPE Tissue kit (Qiagen, Hilden, Germany), and 3) ten biopsies for comparison of the best performing DNA and RNA kits from the two previous steps to the AllPrep® DNA/RNA FFPE kit (Qiagen, Hilden, Germany). .. In order to minimize the risk of variation due to different operators, the same operator performed all extractions with one kit (e.g. all extractions using the RNeasy® FFPE kit).

    Article Title: Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples
    Article Snippet: .. DNA was isolated using the Qiagen QIAamp DNA FFPE Kit according to the manufacturer’s instructions. .. DNA was eluted in 40μl Buffer ATE and concentrations were measured with NanoDrop 2000 (NanoDrop, Wilmington, USA) and Qubit BR kit (Life Technologies, Darmstadt, Germany).

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies
    Article Snippet: .. The macro-dissected tumor area from one tissue section was used for RNA isolation using the RNeasy® FFPE kit (Qiagen, Hilden, Germany) and the tumor area from the second section was used for DNA isolation using the QIAamp® DNA FFPE Tissue kit (Qiagen, Hilden, Germany), following the manufacturers’ instructions. .. Quantity and quality measurements The quantity and purity (A260/A280) of the extracted DNA and RNA was measured using the NanoDrop ND-2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

    Microscopy:

    Article Title: Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples
    Article Snippet: A slide stained with haematoxylin and eosin (H & E) from each block was used to estimate the tumor cell content of the corresponding slices by two investigators (TG and JB) using a double-headed microscope. .. DNA was isolated using the Qiagen QIAamp DNA FFPE Kit according to the manufacturer’s instructions.

    Sequencing:

    Article Title: Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors
    Article Snippet: Sequencing of HPV DNA demonstrated 100% concordance of HPV status. .. Tissue was collected into extraction tubes and processed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany).

    Article Title: A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry
    Article Snippet: The DNA extraction was performed with QIAamp™ DNA FFPE Tissue kit and automated on the QIAcube robot (QIAGEN, Valencia, CA, USA), as previously described and according to the manufacturer’s instructions . .. Firstly, as part of standard clinical practice, we performed the mutation analysis by direct sequencing (the current gold standard).

    Staining:

    Article Title: Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors
    Article Snippet: Paragraph title: Sample collection, p16 staining, and DNA extraction ... Tissue was collected into extraction tubes and processed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany).

    Article Title: Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples
    Article Snippet: A slide stained with haematoxylin and eosin (H & E) from each block was used to estimate the tumor cell content of the corresponding slices by two investigators (TG and JB) using a double-headed microscope. .. DNA was isolated using the Qiagen QIAamp DNA FFPE Kit according to the manufacturer’s instructions.

    Selection:

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum
    Article Snippet: Paragraph title: Tissue selection and DNA isolation ... Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Laser Capture Microdissection:

    Article Title: Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors
    Article Snippet: LCM was carried out on P.A.L.M. .. Tissue was collected into extraction tubes and processed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany).

    Article Title: Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples
    Article Snippet: Suitable blocks were chosen and five 10μm slices were used for DNA extraction without microdissection. .. DNA was isolated using the Qiagen QIAamp DNA FFPE Kit according to the manufacturer’s instructions.

    Incubation:

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum
    Article Snippet: Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.). .. For the FFPE procedure, extraction with xylene was omitted but incubation at 90 °C to reverse formalin crosslinking was performed.

    Article Title: Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method
    Article Snippet: Dedicated Method to FFPE tissue: QIAamp® DNA FFPE Tissue kit (QIAGEN® ) For each sample, ten 15-μm-thick tissue sections were transferred into a 1.5 ml tube. .. In brief, tissue sections were first dewaxed using toluene and lysed under denaturing conditions with proteinase K. Then lysates were incubated at 90°C.

    Lysis:

    Article Title: Critical Issues in Mycobiota Analysis
    Article Snippet: Factors such as residual formalin inhibiting proteinase K activity and omitting complete cell lysis, as well as the presence of PCR inhibitors in the DNA extract might altogether interfere with successful fungal DNA amplification (Coura et al., ; Muñoz-Cadavid et al., ). .. To highlight the influence of pre-analytics on ITS based mycobiota investigations we assessed the performance of DNA extraction from human skin FFPE samples (see Supplementary Table for sample information) with a commercially available kit (QIAamp DNA FFPE tissue kit, Qiagen) reported to be efficient for fungal DNA extraction out of FFPE material (Muñoz-Cadavid et al., ).

    Control Assay:

    Article Title: Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue. A new paradigm in genetic counseling
    Article Snippet: DNA extraction DNA was extracted from three tubes each containing 3 × 15 μ m FFPE tissue sections per sample using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to QIAamp DNA FFPE Tissue Handbook with a few modifications ( www.qiagen.com ). .. DNA extraction DNA was extracted from three tubes each containing 3 × 15 μ m FFPE tissue sections per sample using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to QIAamp DNA FFPE Tissue Handbook with a few modifications ( www.qiagen.com ).

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    Qiagen qiaamp ffpe dna extraction kit
    Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline <t>DNA</t> (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival <t>FFPE</t> tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution
    Qiaamp Ffpe Dna Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1624 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival FFPE tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution

    Journal: NPJ genomic medicine

    Article Title: Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA

    doi: 10.1038/s41525-017-0030-7

    Figure Lengend Snippet: Coverage uniformity of WGS libraries. a Represented is the cumulative proportion of sequencing coverage per cumulative proportion of sequence in whole genome sequencing across normal germline DNA (gDNA) from peripheral blood mononuclear cells, two cfDNA time points, and an archival FFPE tissue biopsy from a metastatic melanoma patient. If coverage was perfectly uniform across the genome coverage the relationship would be linear with gradient one. b Mapped depth of coverage distribution for WGS sequencing runs. The range of the coverage distribution is truncated at 150. Each trace is annotated with the mode of the distribution. c Insert size distribution of sequencing reads for the sequencing runs. The distribution is truncated at 300 base pairs. Each trace is annotated with the mode of the distribution

    Article Snippet: TDNA was extracted using the QIAamp FFPE DNA extraction kit (Qiagen) and eluted into 20 µl buffer EB.

    Techniques: Sequencing, Formalin-fixed Paraffin-Embedded

    Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit

    Journal: BMC Medical Research Methodology

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies

    doi: 10.1186/s12874-018-0628-1

    Figure Lengend Snippet: Bland-Altman plots for investigation of level of agreements between DNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of DNA yield (ng/μl) of samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. b Comparison of purity (A260/A280) of DNA samples extracted with High Pure FFPET DNA Isolation kit and QIAamp® DNA FFPE Tissue kit. c Comparison of DNA yield (ng/μl) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit. d Comparison of purity (A260/A280) of samples extracted with QIAamp® DNA FFPE Tissue kit and AllPrep® DNA/RNA FFPE kit

    Article Snippet: DNA extraction comparative trials In the third comparative trial, Qiagen’s QIAamp® DNA FFPE Tissue and Roche Diagnostic’s High Pure FFPET DNA Isolation kit was investigated.

    Techniques: DNA Extraction, Formalin-fixed Paraffin-Embedded

    Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit

    Journal: BMC Medical Research Methodology

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies

    doi: 10.1186/s12874-018-0628-1

    Figure Lengend Snippet: Bland-Altman plots for investigating the level of agreement between RNA extraction kits. Each plot shows the differences between the two kits against the averages of the two kits. The lines represent the mean differences and upper and lower limits of agreement (LOA, mean differences ±1.96SD). a Comparison of RNA yield (ng/μl) of samples extracted with High Pure FFPE RNA Micro Kit and RNeasy® FFPE kit. b Comparison of purity (A260/A280) of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. c Comparison of RIN-values of samples extracted with High Pure FFPE RNA Micro kit and RNeasy® FFPE kit. d Comparison of RNA yield (ng/μl) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. e Comparison of purity (A260/A280) of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit. f Comparison of RIN-values of samples extracted with RNeasy® FFPE kit and AllPrep® DNA/RNA FFPE kit

    Article Snippet: DNA extraction comparative trials In the third comparative trial, Qiagen’s QIAamp® DNA FFPE Tissue and Roche Diagnostic’s High Pure FFPET DNA Isolation kit was investigated.

    Techniques: RNA Extraction, Formalin-fixed Paraffin-Embedded

    Generation of sample mixes for analytic validation DNA from seven original samples was diluted to generate seven mixes. A. AN3CA and MFE-296 cell lines were mixed 1:1 to create Mix A, which was then mixed 1:1 with HCC827 to create Mix B and once again to create Mix C. B. Two frozen tumor samples and C. two FFPE tumor samples were combined 1:1 and 85:15 to generate mixes D and E (frozen) and F and G (FFPE).

    Journal: Oncotarget

    Article Title: Analytic validation and real-time clinical application of an amplicon-based targeted gene panel for advanced cancer

    doi: 10.18632/oncotarget.20616

    Figure Lengend Snippet: Generation of sample mixes for analytic validation DNA from seven original samples was diluted to generate seven mixes. A. AN3CA and MFE-296 cell lines were mixed 1:1 to create Mix A, which was then mixed 1:1 with HCC827 to create Mix B and once again to create Mix C. B. Two frozen tumor samples and C. two FFPE tumor samples were combined 1:1 and 85:15 to generate mixes D and E (frozen) and F and G (FFPE).

    Article Snippet: DNA extraction and QC DNA was isolated from cell lines and frozen tumors using QiaAMP DNA mini kit (Qiagen, Hilden, Germany), from blood using QiaAMP blood mini kit (Qiagen), and from FFPE tumors using QiaAMP DNA FFPE Tissue kit (Qiagen) per manufacturer's instructions.

    Techniques: Formalin-fixed Paraffin-Embedded