allprep dna rna ffpe kit  (Qiagen)

 
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    Name:
    AllPrep DNA RNA FFPE Kit
    Description:
    For simultaneous purification of genomic DNA and total RNA including small RNAs from formalin fixed paraffin embedded tissue sections Kit contents Qiagen AllPrep DNA RNA FFPE Kit 50 preps FFPE Tissue Sample RNA 14 to 30L DNA 30 to 100L Elution Volume Silica Technology Manual Processing For Simultaneous Purification of Genomic DNA and Total RNA from Formalin fixed Paraffin embedded Tissue Sections Ideal for PCR qPCR Real time RT PCR Microarray Includes 50 RNeasy minElute Spin Columns 50 QIAamp minElute Spin Columns Collection Tubes RNase free Reagents and Buffers Benefits Maximum output with minimal sample consumption Releases DNA RNA without compromising integrity Effectively separates RNA and DNA Comprehensive DNA and RNA analysis of the same FFPE sam
    Catalog Number:
    80234
    Price:
    660
    Category:
    AllPrep DNA RNA FFPE Kit
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    Structured Review

    Qiagen allprep dna rna ffpe kit
    AllPrep DNA RNA FFPE Kit
    For simultaneous purification of genomic DNA and total RNA including small RNAs from formalin fixed paraffin embedded tissue sections Kit contents Qiagen AllPrep DNA RNA FFPE Kit 50 preps FFPE Tissue Sample RNA 14 to 30L DNA 30 to 100L Elution Volume Silica Technology Manual Processing For Simultaneous Purification of Genomic DNA and Total RNA from Formalin fixed Paraffin embedded Tissue Sections Ideal for PCR qPCR Real time RT PCR Microarray Includes 50 RNeasy minElute Spin Columns 50 QIAamp minElute Spin Columns Collection Tubes RNase free Reagents and Buffers Benefits Maximum output with minimal sample consumption Releases DNA RNA without compromising integrity Effectively separates RNA and DNA Comprehensive DNA and RNA analysis of the same FFPE sam
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    allprep dna rna ffpe kit - by Bioz Stars, 2020-07
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    Images

    1) 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

    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

    2) Product Images from "The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss"

    Article Title: The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008642

    UESL display aberrant transcriptional activity of the C19MC region. A novel PEG3/ZIM2 -C19MC fusion is identified. A, Read counts of mapped whole transcriptome show high levels of aberrant transcriptional activity in the C19MC region. Note that the abrupt starting location of transcriptional mapping is in different co-ordinates in different UESL samples, suggestive of sample specific fusional events. The genomic position of the experimentally verified fusion in PATWXD is indicated by the red arrow in panel-A and notably corresponds to the start (5’ end) of transcriptional activity in this sample. Hep3B cell line (hepatocellular carcinoma) and normal liver samples are shown at the bottom for comparison and as expected show negligible amounts of RNA mapping to this non-coding region. B, Targeted DNA sequencing of PATWXD UESL tumor showing abrupt end of read mapping near the C19MC start site (left) as well as in the PEG2/ZIM2 gene locus (right, shared gene region). The reads also mark the position of primers designed for gDNA PCR (one primer at 5’ end of reads and another at 3’end of reads for each locus (therefore one set of primers will form a nested primer set). C, Nested multiplex PCR of PATWXD genomic DNA showing amplicon (~550 bp) including the nested product (~450 bp). D, Paired end Sanger sequencing of ~550 bp product from panel-B showing the PEG3/ZIM2 locus fused to C19MC aberrant transcriptional start site.
    Figure Legend Snippet: UESL display aberrant transcriptional activity of the C19MC region. A novel PEG3/ZIM2 -C19MC fusion is identified. A, Read counts of mapped whole transcriptome show high levels of aberrant transcriptional activity in the C19MC region. Note that the abrupt starting location of transcriptional mapping is in different co-ordinates in different UESL samples, suggestive of sample specific fusional events. The genomic position of the experimentally verified fusion in PATWXD is indicated by the red arrow in panel-A and notably corresponds to the start (5’ end) of transcriptional activity in this sample. Hep3B cell line (hepatocellular carcinoma) and normal liver samples are shown at the bottom for comparison and as expected show negligible amounts of RNA mapping to this non-coding region. B, Targeted DNA sequencing of PATWXD UESL tumor showing abrupt end of read mapping near the C19MC start site (left) as well as in the PEG2/ZIM2 gene locus (right, shared gene region). The reads also mark the position of primers designed for gDNA PCR (one primer at 5’ end of reads and another at 3’end of reads for each locus (therefore one set of primers will form a nested primer set). C, Nested multiplex PCR of PATWXD genomic DNA showing amplicon (~550 bp) including the nested product (~450 bp). D, Paired end Sanger sequencing of ~550 bp product from panel-B showing the PEG3/ZIM2 locus fused to C19MC aberrant transcriptional start site.

    Techniques Used: Activity Assay, DNA Sequencing, Polymerase Chain Reaction, Multiplex Assay, Amplification, Sequencing

    3) Product Images from "Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples"

    Article Title: Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples

    Journal: Toxicological sciences : an official journal of the Society of Toxicology

    doi: 10.1093/toxsci/kfx278

    Effects of formalin, fixation, and demodification on total RNA, amplifiable RNA, and RNA integrity. A) Total RNA yield as assessed by Nanodrop spectrophotometer and Qubit fluorometer. B) RNA integrity number as measured by Bioanalyzer. C) Amplifiable Actb RNA as measured by reverse transcriptase quantitative PCR of three amplicons across the gene body. *The results of a statistical comparison between OH, 18F, and 3F vs. FR with a p-value
    Figure Legend Snippet: Effects of formalin, fixation, and demodification on total RNA, amplifiable RNA, and RNA integrity. A) Total RNA yield as assessed by Nanodrop spectrophotometer and Qubit fluorometer. B) RNA integrity number as measured by Bioanalyzer. C) Amplifiable Actb RNA as measured by reverse transcriptase quantitative PCR of three amplicons across the gene body. *The results of a statistical comparison between OH, 18F, and 3F vs. FR with a p-value

    Techniques Used: Spectrophotometry, Real-time Polymerase Chain Reaction

    4) 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

    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

    5) Product Images from "Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas"

    Article Title: Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0219724

    Comprehensive rendering of multiscale measurements in gliomas. Multiscale modalities depicted include: 1) clinical information (red), 2) IDH1 mutational status (blue), 3) MRI derived variables (green), 4) RNA expression level of genes involved in the “inducing angiogenesis” hallmark (black), and 5) MxIF angiogenesis markers or cell clusters (magenta). The data is binned in low, medium and high categories. Across the treatment-naïve gliomas (a) and the recurrent (post-treatment) glioblastoma* (b) cohorts, it can be observed that IDHmt patients have low angiogenesis according to RNA expression levels and expression of S100A4 and VEGRF. Those subjects also have high fraction of cells in clusters 1 and 2 (low angiogenesis markers), and low fraction of cells in cluster 4 and 5 (high angiogenesis markers ( S8 Fig , cluster profiles of angiogenesis clusters). Moreover, MR Images for the same subjects have lower normalized enhancing cores volumes and measure higher intensities on T1 post contrast. *Recurrent GBM (5 subjects are not shown since they were missing MxIF).
    Figure Legend Snippet: Comprehensive rendering of multiscale measurements in gliomas. Multiscale modalities depicted include: 1) clinical information (red), 2) IDH1 mutational status (blue), 3) MRI derived variables (green), 4) RNA expression level of genes involved in the “inducing angiogenesis” hallmark (black), and 5) MxIF angiogenesis markers or cell clusters (magenta). The data is binned in low, medium and high categories. Across the treatment-naïve gliomas (a) and the recurrent (post-treatment) glioblastoma* (b) cohorts, it can be observed that IDHmt patients have low angiogenesis according to RNA expression levels and expression of S100A4 and VEGRF. Those subjects also have high fraction of cells in clusters 1 and 2 (low angiogenesis markers), and low fraction of cells in cluster 4 and 5 (high angiogenesis markers ( S8 Fig , cluster profiles of angiogenesis clusters). Moreover, MR Images for the same subjects have lower normalized enhancing cores volumes and measure higher intensities on T1 post contrast. *Recurrent GBM (5 subjects are not shown since they were missing MxIF).

    Techniques Used: Magnetic Resonance Imaging, Derivative Assay, RNA Expression, Expressing

    6) Product Images from "Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases"

    Article Title: Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases

    Journal: Nature chemistry

    doi: 10.1038/nchem.2307

    Enhancement in recovery of RNAs from formalin-fixed, paraffin-embedded cell specimens using catalyst 3 (20 mM) as compared with different incubation and isolation conditions. Amplifiable RNA yield is plotted for eight amplicons, and quantity is determined with a standard curve. Lane 1 employs incubation conditions from a commercial kit (Qiagen AllPrep ® DNA/RNA FFPE kit), which uses an 80 °C, 0.25 h incubation step without catalyst (“no cat”) and a spin column for isolation, with the addition of catalyst to these conditions shown in lane 2 (“cat. 3”). Optimized incubation conditions (55 °C, 18 h) followed by a spin column RNA isolation are shown in lanes 3–4. A common literature procedure is shown in lane 5 (“PCI”) 28 . Addition of the catalyst to the optimized incubation conditions results in a ~2 fold increase in detectable RNA, and more substantial increases relative to the catalyst-free commercial kit protocol (see enhancements in red) or the literature protocol. The means of three independent experiments are shown, error bars indicating the standard deviation of variation in the qRT-PCR yield. SC: spin column isolation. PCI: Masuda protocol of phenol-chloroform-isoamyl alcohol extraction followed by heating in buffer. 28 A.U.: arbitrary units. Significance for pairwise comparisons shown was tested using a 1-tailed paired samples t-test. *: P
    Figure Legend Snippet: Enhancement in recovery of RNAs from formalin-fixed, paraffin-embedded cell specimens using catalyst 3 (20 mM) as compared with different incubation and isolation conditions. Amplifiable RNA yield is plotted for eight amplicons, and quantity is determined with a standard curve. Lane 1 employs incubation conditions from a commercial kit (Qiagen AllPrep ® DNA/RNA FFPE kit), which uses an 80 °C, 0.25 h incubation step without catalyst (“no cat”) and a spin column for isolation, with the addition of catalyst to these conditions shown in lane 2 (“cat. 3”). Optimized incubation conditions (55 °C, 18 h) followed by a spin column RNA isolation are shown in lanes 3–4. A common literature procedure is shown in lane 5 (“PCI”) 28 . Addition of the catalyst to the optimized incubation conditions results in a ~2 fold increase in detectable RNA, and more substantial increases relative to the catalyst-free commercial kit protocol (see enhancements in red) or the literature protocol. The means of three independent experiments are shown, error bars indicating the standard deviation of variation in the qRT-PCR yield. SC: spin column isolation. PCI: Masuda protocol of phenol-chloroform-isoamyl alcohol extraction followed by heating in buffer. 28 A.U.: arbitrary units. Significance for pairwise comparisons shown was tested using a 1-tailed paired samples t-test. *: P

    Techniques Used: Formalin-fixed Paraffin-Embedded, Incubation, Isolation, Standard Deviation, Quantitative RT-PCR

    7) Product Images from "A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples"

    Article Title: A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-6166-3

    Genome alignment profiles of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, all the four kits got similar alignment profiles while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng. For FFPE RNA from GM 12878 cell line, the library with TaKaRa kit produced more exon profiles with 10 ng total RNA input
    Figure Legend Snippet: Genome alignment profiles of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, all the four kits got similar alignment profiles while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng. For FFPE RNA from GM 12878 cell line, the library with TaKaRa kit produced more exon profiles with 10 ng total RNA input

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

    Comparison of transcripts quantification in FFPE and FF samples across four kits. High concordance in transcript quantifications were got between FF and FFPE samples using any kit. For either FFPE or FF RNA from GM 12878, the Pearson R between TaKaRa kit and the other three kits were lower and higher similarity was got among KAPA, Vazyme and QIAGEN kits
    Figure Legend Snippet: Comparison of transcripts quantification in FFPE and FF samples across four kits. High concordance in transcript quantifications were got between FF and FFPE samples using any kit. For either FFPE or FF RNA from GM 12878, the Pearson R between TaKaRa kit and the other three kits were lower and higher similarity was got among KAPA, Vazyme and QIAGEN kits

    Techniques Used: Formalin-fixed Paraffin-Embedded

    The distribution of transcripts of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, more low-expressed transcripts were detected in the library of TaKaRa with only 10 ng total RNA input. For FFPE RNA from GM 12878 cell line, similar transcripts were detected while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng
    Figure Legend Snippet: The distribution of transcripts of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, more low-expressed transcripts were detected in the library of TaKaRa with only 10 ng total RNA input. For FFPE RNA from GM 12878 cell line, similar transcripts were detected while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng

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

    8) 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

    9) 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

    10) 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

    11) 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

    12) Product Images from "Multiplex genomic profiling of non-small cell lung cancers from the LETS phase III trial of first-line S-1/carboplatin versus paclitaxel/carboplatin: results of a West Japan Oncology Group study"

    Article Title: Multiplex genomic profiling of non-small cell lung cancers from the LETS phase III trial of first-line S-1/carboplatin versus paclitaxel/carboplatin: results of a West Japan Oncology Group study

    Journal: Oncotarget

    doi:

    CONSORT diagram for the study Of the FFPE specimens obtained from 304 advanced NSCLC patients (54%) enrolled in the LETS study, 9 specimens contained no tumor cells and the remaining 295 specimens were subjected to extraction of DNA and RNA. In addition, 229 FFPE specimens were analyzed for MET amplification by FISH.
    Figure Legend Snippet: CONSORT diagram for the study Of the FFPE specimens obtained from 304 advanced NSCLC patients (54%) enrolled in the LETS study, 9 specimens contained no tumor cells and the remaining 295 specimens were subjected to extraction of DNA and RNA. In addition, 229 FFPE specimens were analyzed for MET amplification by FISH.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Amplification, Fluorescence In Situ Hybridization

    13) Product Images from "Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?"

    Article Title: Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

    Journal: BMC Clinical Pathology

    doi: 10.1186/s12907-016-0039-3

    Investigating the relationship between DNA concentration and the input tissue amount: Geometric mean increase in DNA concentration across 6 different FFPE blocks when combining 5 μm sections in a linear fashion and associated standard error of geomean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE, b Qiagen QIAmp DNA FFPE tissue, c Arcturus PicoPure DNA extraction kit, d Maxwell 16 FFPE Tissue LEV DNA Purification Kit
    Figure Legend Snippet: Investigating the relationship between DNA concentration and the input tissue amount: Geometric mean increase in DNA concentration across 6 different FFPE blocks when combining 5 μm sections in a linear fashion and associated standard error of geomean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE, b Qiagen QIAmp DNA FFPE tissue, c Arcturus PicoPure DNA extraction kit, d Maxwell 16 FFPE Tissue LEV DNA Purification Kit

    Techniques Used: Concentration Assay, Formalin-fixed Paraffin-Embedded, DNA Extraction, DNA Purification

    Investigating the relationship between RNA concentration and the input amount of tissue: Geometric mean increase in RNA concentration ( n = 6) when combining 5 μm sections in a linear fashion and associated standard error of mean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE kit, b Qiagen RNeasy, c Arcturus Paradise plus FFPE RNA isolation kit, d Maxwell 16 LEV RNA FFPE Purification kit
    Figure Legend Snippet: Investigating the relationship between RNA concentration and the input amount of tissue: Geometric mean increase in RNA concentration ( n = 6) when combining 5 μm sections in a linear fashion and associated standard error of mean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE kit, b Qiagen RNeasy, c Arcturus Paradise plus FFPE RNA isolation kit, d Maxwell 16 LEV RNA FFPE Purification kit

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

    14) 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

    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

    15) Product Images from "Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?"

    Article Title: Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

    Journal: BMC Clinical Pathology

    doi: 10.1186/s12907-016-0039-3

    Investigating the relationship between DNA concentration and the input tissue amount: Geometric mean increase in DNA concentration across 6 different FFPE blocks when combining 5 μm sections in a linear fashion and associated standard error of geomean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE, b Qiagen QIAmp DNA FFPE tissue, c Arcturus PicoPure DNA extraction kit, d Maxwell 16 FFPE Tissue LEV DNA Purification Kit
    Figure Legend Snippet: Investigating the relationship between DNA concentration and the input tissue amount: Geometric mean increase in DNA concentration across 6 different FFPE blocks when combining 5 μm sections in a linear fashion and associated standard error of geomean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE, b Qiagen QIAmp DNA FFPE tissue, c Arcturus PicoPure DNA extraction kit, d Maxwell 16 FFPE Tissue LEV DNA Purification Kit

    Techniques Used: Concentration Assay, Formalin-fixed Paraffin-Embedded, DNA Extraction, DNA Purification

    Investigating the relationship between RNA concentration and the input amount of tissue: Geometric mean increase in RNA concentration ( n = 6) when combining 5 μm sections in a linear fashion and associated standard error of mean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE kit, b Qiagen RNeasy, c Arcturus Paradise plus FFPE RNA isolation kit, d Maxwell 16 LEV RNA FFPE Purification kit
    Figure Legend Snippet: Investigating the relationship between RNA concentration and the input amount of tissue: Geometric mean increase in RNA concentration ( n = 6) when combining 5 μm sections in a linear fashion and associated standard error of mean, line of Y = X/5 represents a linear relationship a Qiagen AllPrep DNA/RNA FFPE kit, b Qiagen RNeasy, c Arcturus Paradise plus FFPE RNA isolation kit, d Maxwell 16 LEV RNA FFPE Purification kit

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

    16) 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

    17) Product Images from "Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores"

    Article Title: Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0179732

    Comparison of DNA and RNA yields and quality across labs and sample age. ( A) Bar graph (mean ± SD) comparing the yields of DNA and RNA extracted from 12 FFPE samples (circles) in three independent laboratories, using the AllPrep kit. (B) Correlation plot of DNA and RNA yield from the same 12 samples, as a function of age of sample. Each data point represents the yield for a given sample, extracted at a given laboratory, superimposed on a linear regression line. Correlation of sample age with MS-PCR amplification cycle thresholds (C) or total mRNA counts in a NanoString assay (D), based on a representative set of genes assayed in each case. Each data point represents the Cq value or the total mRNA count for a given sample, extracted at a given laboratory, superimposed on a linear regression line. Detailed data and statistical analyses are presented in the supplementary S2 Table .
    Figure Legend Snippet: Comparison of DNA and RNA yields and quality across labs and sample age. ( A) Bar graph (mean ± SD) comparing the yields of DNA and RNA extracted from 12 FFPE samples (circles) in three independent laboratories, using the AllPrep kit. (B) Correlation plot of DNA and RNA yield from the same 12 samples, as a function of age of sample. Each data point represents the yield for a given sample, extracted at a given laboratory, superimposed on a linear regression line. Correlation of sample age with MS-PCR amplification cycle thresholds (C) or total mRNA counts in a NanoString assay (D), based on a representative set of genes assayed in each case. Each data point represents the Cq value or the total mRNA count for a given sample, extracted at a given laboratory, superimposed on a linear regression line. Detailed data and statistical analyses are presented in the supplementary S2 Table .

    Techniques Used: Formalin-fixed Paraffin-Embedded, Mass Spectrometry, Polymerase Chain Reaction, Amplification

    18) 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

    19) Product Images from "Distinct tumor microenvironments of lytic and blastic bone metastases in prostate cancer patients"

    Article Title: Distinct tumor microenvironments of lytic and blastic bone metastases in prostate cancer patients

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/s40425-019-0753-3

    Gene expression from decalcified FFPE prostate cancer in bone. a 16 FFPE derived RNA samples (6 lytic and 10 blastic) were analyzed on an Agilent Tape Station for concentration and integrity to produce RNA Integrity Scores (RIN). b 3 lytic and 4 blastic samples contained sufficient RNA (25-100 ng) to endure adequate probe coverage of the NanoString Human Immune Oncology 360 gene expression panel. Differential expression revealed a list of significantly upregulated (moving right) and downregulated genes (moving left) in lytic prostate cancer metastases compared to blastic types. c Blastic samples were enriched for JAK-STAT pathway genes while ( d ) Lytic samples were enriched for PI3K-AKT gene expression. e , f Lytic samples based on gene expression demonstrate increased immune cell populations relative to blastic samples. Graphs created using Advanced Analysis module from NanoString nSolver application
    Figure Legend Snippet: Gene expression from decalcified FFPE prostate cancer in bone. a 16 FFPE derived RNA samples (6 lytic and 10 blastic) were analyzed on an Agilent Tape Station for concentration and integrity to produce RNA Integrity Scores (RIN). b 3 lytic and 4 blastic samples contained sufficient RNA (25-100 ng) to endure adequate probe coverage of the NanoString Human Immune Oncology 360 gene expression panel. Differential expression revealed a list of significantly upregulated (moving right) and downregulated genes (moving left) in lytic prostate cancer metastases compared to blastic types. c Blastic samples were enriched for JAK-STAT pathway genes while ( d ) Lytic samples were enriched for PI3K-AKT gene expression. e , f Lytic samples based on gene expression demonstrate increased immune cell populations relative to blastic samples. Graphs created using Advanced Analysis module from NanoString nSolver application

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

    20) Product Images from "The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss"

    Article Title: The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008642

    UESL display aberrant transcriptional activity of the C19MC region. A novel PEG3/ZIM2 -C19MC fusion is identified. A, Read counts of mapped whole transcriptome show high levels of aberrant transcriptional activity in the C19MC region. Note that the abrupt starting location of transcriptional mapping is in different co-ordinates in different UESL samples, suggestive of sample specific fusional events. The genomic position of the experimentally verified fusion in PATWXD is indicated by the red arrow in panel-A and notably corresponds to the start (5’ end) of transcriptional activity in this sample. Hep3B cell line (hepatocellular carcinoma) and normal liver samples are shown at the bottom for comparison and as expected show negligible amounts of RNA mapping to this non-coding region. B, Targeted DNA sequencing of PATWXD UESL tumor showing abrupt end of read mapping near the C19MC start site (left) as well as in the PEG2/ZIM2 gene locus (right, shared gene region). The reads also mark the position of primers designed for gDNA PCR (one primer at 5’ end of reads and another at 3’end of reads for each locus (therefore one set of primers will form a nested primer set). C, Nested multiplex PCR of PATWXD genomic DNA showing amplicon (~550 bp) including the nested product (~450 bp). D, Paired end Sanger sequencing of ~550 bp product from panel-B showing the PEG3/ZIM2 locus fused to C19MC aberrant transcriptional start site.
    Figure Legend Snippet: UESL display aberrant transcriptional activity of the C19MC region. A novel PEG3/ZIM2 -C19MC fusion is identified. A, Read counts of mapped whole transcriptome show high levels of aberrant transcriptional activity in the C19MC region. Note that the abrupt starting location of transcriptional mapping is in different co-ordinates in different UESL samples, suggestive of sample specific fusional events. The genomic position of the experimentally verified fusion in PATWXD is indicated by the red arrow in panel-A and notably corresponds to the start (5’ end) of transcriptional activity in this sample. Hep3B cell line (hepatocellular carcinoma) and normal liver samples are shown at the bottom for comparison and as expected show negligible amounts of RNA mapping to this non-coding region. B, Targeted DNA sequencing of PATWXD UESL tumor showing abrupt end of read mapping near the C19MC start site (left) as well as in the PEG2/ZIM2 gene locus (right, shared gene region). The reads also mark the position of primers designed for gDNA PCR (one primer at 5’ end of reads and another at 3’end of reads for each locus (therefore one set of primers will form a nested primer set). C, Nested multiplex PCR of PATWXD genomic DNA showing amplicon (~550 bp) including the nested product (~450 bp). D, Paired end Sanger sequencing of ~550 bp product from panel-B showing the PEG3/ZIM2 locus fused to C19MC aberrant transcriptional start site.

    Techniques Used: Activity Assay, DNA Sequencing, Polymerase Chain Reaction, Multiplex Assay, Amplification, Sequencing

    Related Articles

    Isolation:

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens
    Article Snippet: .. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. .. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples
    Article Snippet: .. For RNA, the AllPrep DNA/RNA FFPE Kit and RNeasy FFPE Kit from QIAGEN, Agencourt FormaPure Kit from Beckman Coulter and truXTRAC FFPE RNA Kit from Covaris were used. .. For the AllPrep DNA/RNA FFPE Kit, simultaneous extraction of DNA and RNA was done.

    Article Title: Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples
    Article Snippet: .. The QIAamp DNA FFPE Tissue Kit, miRNeasy FFPE Kit and AllPrep DNA/RNA FFPE Kit from QIAGEN have been shown to perform well in comparison with other DNA and RNA protocols in previous studies [ , , ]. .. Equal amount of starting material and a randomized order of the consecutive FFPE block sections were used for all methods of extraction.

    Article Title: Quantity and quality of nucleic acids extracted from archival formalin fixed paraffin embedded prostate biopsies
    Article Snippet: .. In the second trial of comparisons, the RNeasy® FFPE kit was compared to the RNA fraction of the samples extracted with Qiagen’s AllPrep® DNA/RNA FFPE kit. .. There was no evidence of difference in yields (11.4 ng/μl versus 12.6 ng/μl), A260/A280 ratios (1.47 versus 1.50) or CT -values (27.4 versus 28.0) between these two kits, although higher RIN-values were seen for samples extracted with the RNeasy® FFPE kit (2.2 versus 1.2, p = 0.006)(Table ).

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens
    Article Snippet: .. Our analyses demonstrated that the two co-extraction methods tested (optimized TRIzol method (TRI), and Qiagen AllPrep DNA/RNA FFPE kit (QDR)) provided higher yields as well as more reliable material for molecular studies than the separate extraction method (Ambion RecoverAll™ kit (AMB)). .. On one side, the QDR has a short pK digestion (15 minutes), and might be automated, but it might not provide the highest amounts of FFPE DNA and RNA.

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens
    Article Snippet: .. Analysis of 200 ng of genomic DNA recovered from 8, 13, 20, 27 and 31 year-old BBD tissue specimens using the TRIzol-based optimized method (TRI) and the Qiagen AllPrep DNA/RNA FFPE (QDR) kit. .. For each specimen 5× 10 µm sections were used for each method and the total amounts of genomic DNA recovered are displayed below the image of the agarose gel, showing that TRI provides at least twice the amount of DNA than QDR.

    Article Title: Multiplex genomic profiling of non-small cell lung cancers from the LETS phase III trial of first-line S-1/carboplatin versus paclitaxel/carboplatin: results of a West Japan Oncology Group study
    Article Snippet: .. DNA and RNA were purified with the use of an Allprep DNA/RNA FFPE Kit (Qiagen, Valencia, CA). .. The isolated RNA was subjected to reverse transcription with the use of a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

    Article Title: Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases
    Article Snippet: .. RNA recovery from FFPE specimens RNA was extracted from a FFPE Raji cell pellet using either the spin-column-based AllPrep® DNA/RNA FFPE kit (Qiagen), according to the manufacturer’s protocol, or a phenol-chloroform-isoamyl alcohol (PCI) extraction procedure. ..

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens
    Article Snippet: .. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. .. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells.

    Purification:

    Article Title: Multiplex genomic profiling of non-small cell lung cancers from the LETS phase III trial of first-line S-1/carboplatin versus paclitaxel/carboplatin: results of a West Japan Oncology Group study
    Article Snippet: .. DNA and RNA were purified with the use of an Allprep DNA/RNA FFPE Kit (Qiagen, Valencia, CA). .. The isolated RNA was subjected to reverse transcription with the use of a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

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    Qiagen allprep dna rna ffpe kit
    Yield and amplifiability of extracted <t>DNA</t> and <t>RNA.</t> (A) Average total amount of DNA. (B) Average total amount of RNA. (C) Amplifiable DNA quantified with the <t>FFPE</t> 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
    Allprep Dna Rna Ffpe Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0197456

    Figure Lengend 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

    Article Snippet: The QIAamp DNA FFPE Tissue Kit, miRNeasy FFPE Kit and AllPrep DNA/RNA FFPE Kit from QIAGEN have been shown to perform well in comparison with other DNA and RNA protocols in previous studies [ , , ].

    Techniques: Formalin-fixed Paraffin-Embedded, Standard Deviation

    Enhancement in recovery of RNAs from formalin-fixed, paraffin-embedded cell specimens using catalyst 3 (20 mM) as compared with different incubation and isolation conditions. Amplifiable RNA yield is plotted for eight amplicons, and quantity is determined with a standard curve. Lane 1 employs incubation conditions from a commercial kit (Qiagen AllPrep ® DNA/RNA FFPE kit), which uses an 80 °C, 0.25 h incubation step without catalyst (“no cat”) and a spin column for isolation, with the addition of catalyst to these conditions shown in lane 2 (“cat. 3”). Optimized incubation conditions (55 °C, 18 h) followed by a spin column RNA isolation are shown in lanes 3–4. A common literature procedure is shown in lane 5 (“PCI”) 28 . Addition of the catalyst to the optimized incubation conditions results in a ~2 fold increase in detectable RNA, and more substantial increases relative to the catalyst-free commercial kit protocol (see enhancements in red) or the literature protocol. The means of three independent experiments are shown, error bars indicating the standard deviation of variation in the qRT-PCR yield. SC: spin column isolation. PCI: Masuda protocol of phenol-chloroform-isoamyl alcohol extraction followed by heating in buffer. 28 A.U.: arbitrary units. Significance for pairwise comparisons shown was tested using a 1-tailed paired samples t-test. *: P

    Journal: Nature chemistry

    Article Title: Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases

    doi: 10.1038/nchem.2307

    Figure Lengend Snippet: Enhancement in recovery of RNAs from formalin-fixed, paraffin-embedded cell specimens using catalyst 3 (20 mM) as compared with different incubation and isolation conditions. Amplifiable RNA yield is plotted for eight amplicons, and quantity is determined with a standard curve. Lane 1 employs incubation conditions from a commercial kit (Qiagen AllPrep ® DNA/RNA FFPE kit), which uses an 80 °C, 0.25 h incubation step without catalyst (“no cat”) and a spin column for isolation, with the addition of catalyst to these conditions shown in lane 2 (“cat. 3”). Optimized incubation conditions (55 °C, 18 h) followed by a spin column RNA isolation are shown in lanes 3–4. A common literature procedure is shown in lane 5 (“PCI”) 28 . Addition of the catalyst to the optimized incubation conditions results in a ~2 fold increase in detectable RNA, and more substantial increases relative to the catalyst-free commercial kit protocol (see enhancements in red) or the literature protocol. The means of three independent experiments are shown, error bars indicating the standard deviation of variation in the qRT-PCR yield. SC: spin column isolation. PCI: Masuda protocol of phenol-chloroform-isoamyl alcohol extraction followed by heating in buffer. 28 A.U.: arbitrary units. Significance for pairwise comparisons shown was tested using a 1-tailed paired samples t-test. *: P

    Article Snippet: RNA recovery from FFPE specimens RNA was extracted from a FFPE Raji cell pellet using either the spin-column-based AllPrep® DNA/RNA FFPE kit (Qiagen), according to the manufacturer’s protocol, or a phenol-chloroform-isoamyl alcohol (PCI) extraction procedure.

    Techniques: Formalin-fixed Paraffin-Embedded, Incubation, Isolation, Standard Deviation, Quantitative RT-PCR

    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: In the second trial of comparisons, the RNeasy® FFPE kit was compared to the RNA fraction of the samples extracted with Qiagen’s AllPrep® DNA/RNA FFPE kit.

    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: In the second trial of comparisons, the RNeasy® FFPE kit was compared to the RNA fraction of the samples extracted with Qiagen’s AllPrep® DNA/RNA FFPE kit.

    Techniques: RNA Extraction, Formalin-fixed Paraffin-Embedded