cfdna  (Qiagen)

 
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    Name:
    QIAamp Circulating Nucleic Acid Kit
    Description:
    For isolation of free circulating DNA and RNA from human plasma or serum Kit contents Qiagen QIAamp Circulating Nucleic Acid Kit 50 preps 1 to 5mL Sample 20 to 150L Elution Volume Serum Plasma Sample Free circulating DNA RNA and miRNA Viral DNA Viral RNA Purification QIAamp Mini Column Format Silica Technology For Isolation of Free circulating DNA and RNA from Human Plasma or Serum Includes QIAamp Mini Columns 20mL Tube Extenders Qiagen Proteinase K Carrier RNA Buffers VacConnectors and 1 5mL and 2mL Collection Tubes Benefits Concentration of nucleic acids with high input and low elution volumes Efficient recovery of fragmented DNA and RNA No organic extraction or ethanol precipitation Removal of contaminants and inhibitors
    Catalog Number:
    55114
    Price:
    1091
    Category:
    QIAamp Circulating Nucleic Acid Kit
    		Buy from Supplier

    Structured Review

    Qiagen cfdna
    QIAamp Circulating Nucleic Acid Kit
    For isolation of free circulating DNA and RNA from human plasma or serum Kit contents Qiagen QIAamp Circulating Nucleic Acid Kit 50 preps 1 to 5mL Sample 20 to 150L Elution Volume Serum Plasma Sample Free circulating DNA RNA and miRNA Viral DNA Viral RNA Purification QIAamp Mini Column Format Silica Technology For Isolation of Free circulating DNA and RNA from Human Plasma or Serum Includes QIAamp Mini Columns 20mL Tube Extenders Qiagen Proteinase K Carrier RNA Buffers VacConnectors and 1 5mL and 2mL Collection Tubes Benefits Concentration of nucleic acids with high input and low elution volumes Efficient recovery of fragmented DNA and RNA No organic extraction or ethanol precipitation Removal of contaminants and inhibitors
    https://www.bioz.com/result/cfdna/product/Qiagen
    Average 92 stars, based on 37068 article reviews
    Price from $9.99 to $1999.99
    cfdna - by Bioz Stars, 2020-09
    92/100 stars

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    Images

    1) Product Images from "Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA"

    Article Title: Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

    Journal: bioRxiv

    doi: 10.1101/2020.01.15.907022

    (A) CfDNA concentrations for 28 cases and 25 controls (B) CelFiE decomposition estimates for 16 ALS patients (light blue) and 16 controls (dark blue).
    Figure Legend Snippet: (A) CfDNA concentrations for 28 cases and 25 controls (B) CelFiE decomposition estimates for 16 ALS patients (light blue) and 16 controls (dark blue).

    Techniques Used:

    2) Product Images from "Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA"

    Article Title: Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

    Journal: bioRxiv

    doi: 10.1101/2020.01.15.907022

    (A) CfDNA concentrations for 28 cases and 25 controls (B) CelFiE decomposition estimates for 16 ALS patients (light blue) and 16 controls (dark blue).
    Figure Legend Snippet: (A) CfDNA concentrations for 28 cases and 25 controls (B) CelFiE decomposition estimates for 16 ALS patients (light blue) and 16 controls (dark blue).

    Techniques Used:

    3) Product Images from "Targeted Sequencing of Genomic Repeat Regions Detects Circulating Cell-free Echinococcus DNA"

    Article Title: Targeted Sequencing of Genomic Repeat Regions Detects Circulating Cell-free Echinococcus DNA

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0008147

    Profiles of cfDNA extracted from intact hydatid cysts. (A) Size of cfDNA from HCF samples analyzed on a 2100 bioanalyzer. Molecular weight (bp) is indicated (left lane). Green lines:15bp. Purple lines: 1500bp. (B) Profile of the cfDNA sequencing reads mapped to the E . granulosus genome (ASM52419v1) and human genome (hg38).
    Figure Legend Snippet: Profiles of cfDNA extracted from intact hydatid cysts. (A) Size of cfDNA from HCF samples analyzed on a 2100 bioanalyzer. Molecular weight (bp) is indicated (left lane). Green lines:15bp. Purple lines: 1500bp. (B) Profile of the cfDNA sequencing reads mapped to the E . granulosus genome (ASM52419v1) and human genome (hg38).

    Techniques Used: Molecular Weight, Sequencing

    4) Product Images from "Selection and evaluation of an efficient method for the recovery of viral nucleic acids from complex biologicals"

    Article Title: Selection and evaluation of an efficient method for the recovery of viral nucleic acids from complex biologicals

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-018-0067-3

    Efficiency of separate extraction of DNA and RNA when compared to total nucleic acid extraction using QIAamp® MinElute® Virus Spin kit. DNA and RNA were extracted separately and compared to the total nucleic acid extracted using the QIAamp® MinElute® Virus Spin kit, using qPCR. RNA extraction using the Qiagen RNeasy® Mini kit did not enhance the detection of the two single-stranded RNA viruses (FeLV and RSV), whereas the RNase A digestion added to the Wako DNA Extractor® kit greatly enriched for double-stranded nucleic acid (EBV and Reo3)
    Figure Legend Snippet: Efficiency of separate extraction of DNA and RNA when compared to total nucleic acid extraction using QIAamp® MinElute® Virus Spin kit. DNA and RNA were extracted separately and compared to the total nucleic acid extracted using the QIAamp® MinElute® Virus Spin kit, using qPCR. RNA extraction using the Qiagen RNeasy® Mini kit did not enhance the detection of the two single-stranded RNA viruses (FeLV and RSV), whereas the RNase A digestion added to the Wako DNA Extractor® kit greatly enriched for double-stranded nucleic acid (EBV and Reo3)

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

    5) Product Images from "Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer"

    Article Title: Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.02.010

    The study design and single-strand library preparation of cfDNA in conception. cfDNA fragments were first denatured into single-strand DNA (ssDNA) fragments. Then, the ssDNA fragments were ligated with a unique molecular identifier. The pre-library was enriched using hybridization and magnetic-beads capture and then subjected to high-throughput sequencing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: The study design and single-strand library preparation of cfDNA in conception. cfDNA fragments were first denatured into single-strand DNA (ssDNA) fragments. Then, the ssDNA fragments were ligated with a unique molecular identifier. The pre-library was enriched using hybridization and magnetic-beads capture and then subjected to high-throughput sequencing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Hybridization, Magnetic Beads, Next-Generation Sequencing

    Ultra-short fragments containing the KRAS hotspot alleles in patients with pancreatic cancer. (a) Comprehensive view of fragment lengths bearing KRAS mutated and wild-type alleles derived from cell-free DNA deep-sequencing in patients with pancreatic cancer ( n = 78). (b) The fragment length of cfDNA bearing mutant KRAS alleles tended to be significantly shorter compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (n = 78). The fragments bearing KRAS mutant alleles tended to be enriched in the region below 100 bp in patients (c) PC076 (stage II) and (d) PC092 (IPMN). (e) The distribution of KRAS mutated fragments included a large proportion of overlapping fragment sizes with wild-type KRAS fragments in advanced patients (PC152). (f) There was considerable discrepancy across IPMN ( n = 10), early-stage ( n = 42) and advanced ( n = 26) pancreatic cancer in terms of the length of KRAS mutated fragments by densitometry. (g) Violin plots representing the length of KRAS mutated fragments across different disease stages. (h) The ability of library preparation to enrich shorter fragments has a great impact on the detection of KRAS hotspot mutations in plasma (blue line), and the mutated-to-wild-type fraction of fragments bearing KRAS alleles reached the highest in the interval of 60–100 bp and declined sharply thereafter (red line). (i) Ultra-short fragments (
    Figure Legend Snippet: Ultra-short fragments containing the KRAS hotspot alleles in patients with pancreatic cancer. (a) Comprehensive view of fragment lengths bearing KRAS mutated and wild-type alleles derived from cell-free DNA deep-sequencing in patients with pancreatic cancer ( n = 78). (b) The fragment length of cfDNA bearing mutant KRAS alleles tended to be significantly shorter compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (n = 78). The fragments bearing KRAS mutant alleles tended to be enriched in the region below 100 bp in patients (c) PC076 (stage II) and (d) PC092 (IPMN). (e) The distribution of KRAS mutated fragments included a large proportion of overlapping fragment sizes with wild-type KRAS fragments in advanced patients (PC152). (f) There was considerable discrepancy across IPMN ( n = 10), early-stage ( n = 42) and advanced ( n = 26) pancreatic cancer in terms of the length of KRAS mutated fragments by densitometry. (g) Violin plots representing the length of KRAS mutated fragments across different disease stages. (h) The ability of library preparation to enrich shorter fragments has a great impact on the detection of KRAS hotspot mutations in plasma (blue line), and the mutated-to-wild-type fraction of fragments bearing KRAS alleles reached the highest in the interval of 60–100 bp and declined sharply thereafter (red line). (i) Ultra-short fragments (

    Techniques Used: Derivative Assay, Sequencing, Mutagenesis

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

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21256

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

    Techniques Used: Concentration Assay

    7) Product Images from "T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids"

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

    Journal: Scientific Reports

    doi: 10.1038/srep40767

    Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification, Generated

    Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction

    TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Amplification

    Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

    8) Product Images from "Innovative methodology for the identification of soluble biomarkers in fresh tissues"

    Article Title: Innovative methodology for the identification of soluble biomarkers in fresh tissues

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24366

    EXPEL extruded fluid contains high quality tumor DNA (tDNA) that is exploitable for genetic profiling (A) Left panel, equivalent yield of tDNA was obtained from FFPE sections and matched EXPEL fluids of CRC primary lesions (n=10) and CRC-liver metastatic lesions (n=10). The quality of the DNA was assessed on the same extracts. The ratios of long 129bp (middle panel) and 305bp (right panel) to short amplified fragments (41bp) indicated that EXPEL tDNA presented with significantly higher amounts of long fragments in comparison with the FFPE tDNA. Dot plots show the mean ± SEM. Statistical significance was calculated using Wilcoxon paired test ( * p
    Figure Legend Snippet: EXPEL extruded fluid contains high quality tumor DNA (tDNA) that is exploitable for genetic profiling (A) Left panel, equivalent yield of tDNA was obtained from FFPE sections and matched EXPEL fluids of CRC primary lesions (n=10) and CRC-liver metastatic lesions (n=10). The quality of the DNA was assessed on the same extracts. The ratios of long 129bp (middle panel) and 305bp (right panel) to short amplified fragments (41bp) indicated that EXPEL tDNA presented with significantly higher amounts of long fragments in comparison with the FFPE tDNA. Dot plots show the mean ± SEM. Statistical significance was calculated using Wilcoxon paired test ( * p

    Techniques Used: Formalin-fixed Paraffin-Embedded, Amplification

    9) Product Images from "Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors"

    Article Title: Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21982

    Average cfDNA yield (ng/mL) in different solid tumor types studied cfDNA extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay cfDNA yield (ng/mL). n represent the number tested for each tumor type. * one sample excluded from graph due to high cfDNA concentration (769 ng/mL).
    Figure Legend Snippet: Average cfDNA yield (ng/mL) in different solid tumor types studied cfDNA extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay cfDNA yield (ng/mL). n represent the number tested for each tumor type. * one sample excluded from graph due to high cfDNA concentration (769 ng/mL).

    Techniques Used: Concentration Assay

    10) Product Images from "Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer ☆"

    Article Title: Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer ☆

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.02.010

    The study design and single-strand library preparation of cfDNA in conception. cfDNA fragments were first denatured into single-strand DNA (ssDNA) fragments. Then, the ssDNA fragments were ligated with a unique molecular identifier. The pre-library was enriched using hybridization and magnetic-beads capture and then subjected to high-throughput sequencing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: The study design and single-strand library preparation of cfDNA in conception. cfDNA fragments were first denatured into single-strand DNA (ssDNA) fragments. Then, the ssDNA fragments were ligated with a unique molecular identifier. The pre-library was enriched using hybridization and magnetic-beads capture and then subjected to high-throughput sequencing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Hybridization, Magnetic Beads, Next-Generation Sequencing

    Ultra-short fragments containing the KRAS hotspot alleles in patients with pancreatic cancer. (a) Comprehensive view of fragment lengths bearing KRAS mutated and wild-type alleles derived from cell-free DNA deep-sequencing in patients with pancreatic cancer ( n = 78). (b) The fragment length of cfDNA bearing mutant KRAS alleles tended to be significantly shorter compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (n = 78). The fragments bearing KRAS mutant alleles tended to be enriched in the region below 100 bp in patients (c) PC076 (stage II) and (d) PC092 (IPMN). (e) The distribution of KRAS mutated fragments included a large proportion of overlapping fragment sizes with wild-type KRAS fragments in advanced patients (PC152). (f) There was considerable discrepancy across IPMN ( n = 10), early-stage ( n = 42) and advanced ( n = 26) pancreatic cancer in terms of the length of KRAS mutated fragments by densitometry. (g) Violin plots representing the length of KRAS mutated fragments across different disease stages. (h) The ability of library preparation to enrich shorter fragments has a great impact on the detection of KRAS hotspot mutations in plasma (blue line), and the mutated-to-wild-type fraction of fragments bearing KRAS alleles reached the highest in the interval of 60–100 bp and declined sharply thereafter (red line). (i) Ultra-short fragments (
    Figure Legend Snippet: Ultra-short fragments containing the KRAS hotspot alleles in patients with pancreatic cancer. (a) Comprehensive view of fragment lengths bearing KRAS mutated and wild-type alleles derived from cell-free DNA deep-sequencing in patients with pancreatic cancer ( n = 78). (b) The fragment length of cfDNA bearing mutant KRAS alleles tended to be significantly shorter compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (n = 78). The fragments bearing KRAS mutant alleles tended to be enriched in the region below 100 bp in patients (c) PC076 (stage II) and (d) PC092 (IPMN). (e) The distribution of KRAS mutated fragments included a large proportion of overlapping fragment sizes with wild-type KRAS fragments in advanced patients (PC152). (f) There was considerable discrepancy across IPMN ( n = 10), early-stage ( n = 42) and advanced ( n = 26) pancreatic cancer in terms of the length of KRAS mutated fragments by densitometry. (g) Violin plots representing the length of KRAS mutated fragments across different disease stages. (h) The ability of library preparation to enrich shorter fragments has a great impact on the detection of KRAS hotspot mutations in plasma (blue line), and the mutated-to-wild-type fraction of fragments bearing KRAS alleles reached the highest in the interval of 60–100 bp and declined sharply thereafter (red line). (i) Ultra-short fragments (

    Techniques Used: Derivative Assay, Sequencing, Mutagenesis

    11) Product Images from "Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification"

    Article Title: Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-014-7835-3

    Assessment of cell-free DNA (cfDNA) yield using four extraction methods. The mean yield ± one standard deviation from replicate extractions using plasma pool A (i) (with or without ADH ) performed with the QIAamp circulating nucleic acid ( CNA ), NucleoSpin Plasma XS ( NS ) and FitAmp plasma/serum DNA isolation ( FA ) kits ( n = 10) and the QIAamp DNA blood mini ( DBM ) kit ( n = 9) is displayed relative to the mean yield of the DBM kit. The yield of cfDNA was quantified by quantitative PCR (qPCR) assays to TERT and ALUJ
    Figure Legend Snippet: Assessment of cell-free DNA (cfDNA) yield using four extraction methods. The mean yield ± one standard deviation from replicate extractions using plasma pool A (i) (with or without ADH ) performed with the QIAamp circulating nucleic acid ( CNA ), NucleoSpin Plasma XS ( NS ) and FitAmp plasma/serum DNA isolation ( FA ) kits ( n = 10) and the QIAamp DNA blood mini ( DBM ) kit ( n = 9) is displayed relative to the mean yield of the DBM kit. The yield of cfDNA was quantified by quantitative PCR (qPCR) assays to TERT and ALUJ

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

    12) Product Images from "Innovative methodology for the identification of soluble biomarkers in fresh tissues"

    Article Title: Innovative methodology for the identification of soluble biomarkers in fresh tissues

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24366

    EXPEL extruded fluid contains high quality tumor DNA (tDNA) that is exploitable for genetic profiling (A) Left panel, equivalent yield of tDNA was obtained from FFPE sections and matched EXPEL fluids of CRC primary lesions (n=10) and CRC-liver metastatic lesions (n=10). The quality of the DNA was assessed on the same extracts. The ratios of long 129bp (middle panel) and 305bp (right panel) to short amplified fragments (41bp) indicated that EXPEL tDNA presented with significantly higher amounts of long fragments in comparison with the FFPE tDNA. Dot plots show the mean ± SEM. Statistical significance was calculated using Wilcoxon paired test ( * p
    Figure Legend Snippet: EXPEL extruded fluid contains high quality tumor DNA (tDNA) that is exploitable for genetic profiling (A) Left panel, equivalent yield of tDNA was obtained from FFPE sections and matched EXPEL fluids of CRC primary lesions (n=10) and CRC-liver metastatic lesions (n=10). The quality of the DNA was assessed on the same extracts. The ratios of long 129bp (middle panel) and 305bp (right panel) to short amplified fragments (41bp) indicated that EXPEL tDNA presented with significantly higher amounts of long fragments in comparison with the FFPE tDNA. Dot plots show the mean ± SEM. Statistical significance was calculated using Wilcoxon paired test ( * p

    Techniques Used: Formalin-fixed Paraffin-Embedded, Amplification

    13) Product Images from "Efficient treatment of a metastatic melanoma patient with a combination of BRAF and MEK inhibitors based on circulating tumor DNA analysis: a case report"

    Article Title: Efficient treatment of a metastatic melanoma patient with a combination of BRAF and MEK inhibitors based on circulating tumor DNA analysis: a case report

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2650-5

    Detection of BRAF V600E mutations in the patient’s plasma. Plasma was collected every day when the patient was in the hospital (9 days), and at each clinical evaluation (after 4 and 8 weeks of treatment). DNA was extracted from plasma (2 mL) using the QIAamp circulating nucleic acid kit (Qiagen). BRAF V600E mutations were detected and quantified by digital PCR using the QuantStudio 3D system and a specific BRAF V600E probe (Thermo Fischer, Courtaboeuf, France)
    Figure Legend Snippet: Detection of BRAF V600E mutations in the patient’s plasma. Plasma was collected every day when the patient was in the hospital (9 days), and at each clinical evaluation (after 4 and 8 weeks of treatment). DNA was extracted from plasma (2 mL) using the QIAamp circulating nucleic acid kit (Qiagen). BRAF V600E mutations were detected and quantified by digital PCR using the QuantStudio 3D system and a specific BRAF V600E probe (Thermo Fischer, Courtaboeuf, France)

    Techniques Used: Digital PCR

    14) Product Images from "T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids"

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

    Journal: Scientific Reports

    doi: 10.1038/srep40767

    Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification, Generated

    Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction

    TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Amplification

    Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

    15) Product Images from "Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions"

    Article Title: Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

    Journal: Diagnostics

    doi: 10.3390/diagnostics10040234

    Comparison of cfDNA isolation efficiencies by four different extraction methods. ( A ) Comparison of the cfDNA yield expressed as the amount of DNA obtained from 1 mL of urine. ( B ) Comparison of the degree of contamination by cellular genomic DNA. The degree of contamination was assessed as the ratio of cfDNA (50–300 bp) to high molecular weight (HMW) DNA ( > 1 kb) (QC; QIAamp Circulating Nucleic Acid Kit, MM; MagMAX™ Cell-Free DNA Isolation Kit, NU; Norgen Urine Cell-Free Circulating DNA Purification Midi Kit, ZQ; Zymo research Quick-DNA™ Urine Kit).
    Figure Legend Snippet: Comparison of cfDNA isolation efficiencies by four different extraction methods. ( A ) Comparison of the cfDNA yield expressed as the amount of DNA obtained from 1 mL of urine. ( B ) Comparison of the degree of contamination by cellular genomic DNA. The degree of contamination was assessed as the ratio of cfDNA (50–300 bp) to high molecular weight (HMW) DNA ( > 1 kb) (QC; QIAamp Circulating Nucleic Acid Kit, MM; MagMAX™ Cell-Free DNA Isolation Kit, NU; Norgen Urine Cell-Free Circulating DNA Purification Midi Kit, ZQ; Zymo research Quick-DNA™ Urine Kit).

    Techniques Used: Isolation, Molecular Weight, DNA Extraction, DNA Purification

    Representative electropherograms visualized on the Bioanalyzer using high sensitivity DNA chips. Electropherograms show the cell-free DNA (cfDNA) fragment distributions obtained by four different extraction methods using the same sample. The cfDNA concentration was calculated for fractions in the size range of 50–300 bp (QC; QIAamp Circulating Nucleic Acid Kit, MM; MagMAX™ Cell-Free DNA Isolation Kit, NU; Norgen Urine Cell-Free Circulating DNA Purification Midi Kit, ZQ; Zymo research Quick-DNA™ Urine Kit).
    Figure Legend Snippet: Representative electropherograms visualized on the Bioanalyzer using high sensitivity DNA chips. Electropherograms show the cell-free DNA (cfDNA) fragment distributions obtained by four different extraction methods using the same sample. The cfDNA concentration was calculated for fractions in the size range of 50–300 bp (QC; QIAamp Circulating Nucleic Acid Kit, MM; MagMAX™ Cell-Free DNA Isolation Kit, NU; Norgen Urine Cell-Free Circulating DNA Purification Midi Kit, ZQ; Zymo research Quick-DNA™ Urine Kit).

    Techniques Used: Concentration Assay, DNA Extraction, DNA Purification

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150197

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

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

    17) Product Images from "Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer ☆"

    Article Title: Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer ☆

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.02.010

    The study design and single-strand library preparation of cfDNA in conception. cfDNA fragments were first denatured into single-strand DNA (ssDNA) fragments. Then, the ssDNA fragments were ligated with a unique molecular identifier. The pre-library was enriched using hybridization and magnetic-beads capture and then subjected to high-throughput sequencing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: The study design and single-strand library preparation of cfDNA in conception. cfDNA fragments were first denatured into single-strand DNA (ssDNA) fragments. Then, the ssDNA fragments were ligated with a unique molecular identifier. The pre-library was enriched using hybridization and magnetic-beads capture and then subjected to high-throughput sequencing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Hybridization, Magnetic Beads, Next-Generation Sequencing

    Ultra-short fragments containing the KRAS hotspot alleles in patients with pancreatic cancer. (a) Comprehensive view of fragment lengths bearing KRAS mutated and wild-type alleles derived from cell-free DNA deep-sequencing in patients with pancreatic cancer ( n = 78). (b) The fragment length of cfDNA bearing mutant KRAS alleles tended to be significantly shorter compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (n = 78). The fragments bearing KRAS mutant alleles tended to be enriched in the region below 100 bp in patients (c) PC076 (stage II) and (d) PC092 (IPMN). (e) The distribution of KRAS mutated fragments included a large proportion of overlapping fragment sizes with wild-type KRAS fragments in advanced patients (PC152). (f) There was considerable discrepancy across IPMN ( n = 10), early-stage ( n = 42) and advanced ( n = 26) pancreatic cancer in terms of the length of KRAS mutated fragments by densitometry. (g) Violin plots representing the length of KRAS mutated fragments across different disease stages. (h) The ability of library preparation to enrich shorter fragments has a great impact on the detection of KRAS hotspot mutations in plasma (blue line), and the mutated-to-wild-type fraction of fragments bearing KRAS alleles reached the highest in the interval of 60–100 bp and declined sharply thereafter (red line). (i) Ultra-short fragments (
    Figure Legend Snippet: Ultra-short fragments containing the KRAS hotspot alleles in patients with pancreatic cancer. (a) Comprehensive view of fragment lengths bearing KRAS mutated and wild-type alleles derived from cell-free DNA deep-sequencing in patients with pancreatic cancer ( n = 78). (b) The fragment length of cfDNA bearing mutant KRAS alleles tended to be significantly shorter compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (n = 78). The fragments bearing KRAS mutant alleles tended to be enriched in the region below 100 bp in patients (c) PC076 (stage II) and (d) PC092 (IPMN). (e) The distribution of KRAS mutated fragments included a large proportion of overlapping fragment sizes with wild-type KRAS fragments in advanced patients (PC152). (f) There was considerable discrepancy across IPMN ( n = 10), early-stage ( n = 42) and advanced ( n = 26) pancreatic cancer in terms of the length of KRAS mutated fragments by densitometry. (g) Violin plots representing the length of KRAS mutated fragments across different disease stages. (h) The ability of library preparation to enrich shorter fragments has a great impact on the detection of KRAS hotspot mutations in plasma (blue line), and the mutated-to-wild-type fraction of fragments bearing KRAS alleles reached the highest in the interval of 60–100 bp and declined sharply thereafter (red line). (i) Ultra-short fragments (

    Techniques Used: Derivative Assay, Sequencing, Mutagenesis

    18) Product Images from "Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid"

    Article Title: Evaluating Cancer of the Central Nervous System Through Next-Generation Sequencing of Cerebrospinal Fluid

    Journal: Journal of Clinical Oncology

    doi: 10.1200/JCO.2016.66.6487

    Tumor evolution in patients with primary brain tumors. (A) Spatial and temporal heterogeneity among samples obtained at diagnosis, at recurrence, and from cerebrospinal fluid (CSF) in patient 42 with recurrent glioblastoma. CSF cell-free DNA harbors a PTEN R130* mutation (variant allele frequency, 0.25), whereas resection 2 harbors a PIK3CA H1047R mutation (variant allele frequency, 0.441). (B) CSF molecular profile for patient 45 with anaplastic oligodendroglioma contains the IDH1 R132H mutation and 1p/19q deletion found in tissue resection 2 as well as 454 nonsilent somatic mutations. Four hundred forty-eight SNVs represent C > T/G > A mutations that demonstrate TMZ-induced mutagenesis. Carbo, carboplatin; CCNU, lomustine; rhuMAB VEGF, bevacuzumab; RT, radiotherapy; SNV, single nucleotide variant; TMZ, temozolomide.
    Figure Legend Snippet: Tumor evolution in patients with primary brain tumors. (A) Spatial and temporal heterogeneity among samples obtained at diagnosis, at recurrence, and from cerebrospinal fluid (CSF) in patient 42 with recurrent glioblastoma. CSF cell-free DNA harbors a PTEN R130* mutation (variant allele frequency, 0.25), whereas resection 2 harbors a PIK3CA H1047R mutation (variant allele frequency, 0.441). (B) CSF molecular profile for patient 45 with anaplastic oligodendroglioma contains the IDH1 R132H mutation and 1p/19q deletion found in tissue resection 2 as well as 454 nonsilent somatic mutations. Four hundred forty-eight SNVs represent C > T/G > A mutations that demonstrate TMZ-induced mutagenesis. Carbo, carboplatin; CCNU, lomustine; rhuMAB VEGF, bevacuzumab; RT, radiotherapy; SNV, single nucleotide variant; TMZ, temozolomide.

    Techniques Used: Mutagenesis, Variant Assay

    Comparison of tumor-derived DNA from cerebrospinal fluid (CSF) cell pellet and supernatant. (A) Schematic of separation of CSF pellet and supernatant. Cellular DNA is isolated from the pellet, and cell-free DNA (cfDNA) is isolated from the supernatant. (B) Variant allele frequencies for known mutations in CSF cfDNA and pellet DNA. (C) Log2 ratios of normalized sequence coverage for target exons in CSF cfDNA and pellet DNA for patient 8. Greater than 10-fold amplification of HER2 was observed in CSF cfDNA, whereas HER2 amplification was barely detectable in pellet DNA. (D) Evidence of EML4-ALK gene fusion in CSF cfDNA and pellet DNA for patient 6. Read pairs supporting the fusion (red) were visualized by using the Integrative Genomics Viewer. Pt, patient ID.
    Figure Legend Snippet: Comparison of tumor-derived DNA from cerebrospinal fluid (CSF) cell pellet and supernatant. (A) Schematic of separation of CSF pellet and supernatant. Cellular DNA is isolated from the pellet, and cell-free DNA (cfDNA) is isolated from the supernatant. (B) Variant allele frequencies for known mutations in CSF cfDNA and pellet DNA. (C) Log2 ratios of normalized sequence coverage for target exons in CSF cfDNA and pellet DNA for patient 8. Greater than 10-fold amplification of HER2 was observed in CSF cfDNA, whereas HER2 amplification was barely detectable in pellet DNA. (D) Evidence of EML4-ALK gene fusion in CSF cfDNA and pellet DNA for patient 6. Read pairs supporting the fusion (red) were visualized by using the Integrative Genomics Viewer. Pt, patient ID.

    Techniques Used: Derivative Assay, Isolation, Variant Assay, Sequencing, Amplification

    Drug-resistance mutations in patients whose central nervous system (CNS) disease progresses during kinase inhibitor therapy. (A) Summary of genomic profiling results from cerebrospinal fluid (CSF) and other tumor sites in patients in whom progressive CNS disease developed during treatment with the indicated kinase inhibitors. (B) Disease timeline and brain magnet resonance images (MRIs) from a patient with EGFR -mutant NSCLC (patient 3) who presented with leptomeningeal metastasis (baseline MRI, arrows), responded to erlotinib (follow-up MRI at 26 months), was found to have a secondary EGFR mutation (T790M) in a bone metastasis, and developed progressive CNS disease (brain MRIs at 32 and 35 months) that did not respond to second-generation EGFR TKI or pulse erlotinib. CSF cell-free DNA (cfDNA) identified an EGFR T790M mutation. (C) Disease timeline and brain MRIs from a patient with EGFR -mutant NSCLC (patient 4) who presented with brain metastases (baseline MRI), responded to erlotinib (follow-up brain MRI at 2 months and brain CT scan at 9 months), and later developed progressive brain metastases. Molecular profiling of the recurrent lung tumor showed a secondary EGFR mutation (T790M), whereas CSF cfDNA identified an activating KRAS mutation (and the absence of T790M). Sequenom mass spectrometry genotyping was performed for specific mutations in eight genes: AKT1 , BRAF , EGFR , ERBB2 , KRAS , MEK1 ( MAP2K1 ), NRAS , and PIK3CA . ALK, anaplastic lymphoma kinase; AMP, amplification; BrCa, breast cancer; CT, computed tomography; del, deletion; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; IMPACT, Integrated Molecular Profiling of Actionable Cancer Targets; ND, not determined; NSCLC, non–small-cell lung cancer; PCR, polymerase chain reaction; pert, pertuzumab; T-DM1, trastuzumab emtansine; TKI, tyrosine kinase inhibitor; trast, trastuzumab; WBRT, whole-brain radiotherapy.
    Figure Legend Snippet: Drug-resistance mutations in patients whose central nervous system (CNS) disease progresses during kinase inhibitor therapy. (A) Summary of genomic profiling results from cerebrospinal fluid (CSF) and other tumor sites in patients in whom progressive CNS disease developed during treatment with the indicated kinase inhibitors. (B) Disease timeline and brain magnet resonance images (MRIs) from a patient with EGFR -mutant NSCLC (patient 3) who presented with leptomeningeal metastasis (baseline MRI, arrows), responded to erlotinib (follow-up MRI at 26 months), was found to have a secondary EGFR mutation (T790M) in a bone metastasis, and developed progressive CNS disease (brain MRIs at 32 and 35 months) that did not respond to second-generation EGFR TKI or pulse erlotinib. CSF cell-free DNA (cfDNA) identified an EGFR T790M mutation. (C) Disease timeline and brain MRIs from a patient with EGFR -mutant NSCLC (patient 4) who presented with brain metastases (baseline MRI), responded to erlotinib (follow-up brain MRI at 2 months and brain CT scan at 9 months), and later developed progressive brain metastases. Molecular profiling of the recurrent lung tumor showed a secondary EGFR mutation (T790M), whereas CSF cfDNA identified an activating KRAS mutation (and the absence of T790M). Sequenom mass spectrometry genotyping was performed for specific mutations in eight genes: AKT1 , BRAF , EGFR , ERBB2 , KRAS , MEK1 ( MAP2K1 ), NRAS , and PIK3CA . ALK, anaplastic lymphoma kinase; AMP, amplification; BrCa, breast cancer; CT, computed tomography; del, deletion; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; IMPACT, Integrated Molecular Profiling of Actionable Cancer Targets; ND, not determined; NSCLC, non–small-cell lung cancer; PCR, polymerase chain reaction; pert, pertuzumab; T-DM1, trastuzumab emtansine; TKI, tyrosine kinase inhibitor; trast, trastuzumab; WBRT, whole-brain radiotherapy.

    Techniques Used: Mutagenesis, Magnetic Resonance Imaging, Computed Tomography, Mass Spectrometry, Amplification, Fluorescence In Situ Hybridization, In Situ Hybridization, Immunohistochemistry, Polymerase Chain Reaction

    19) Product Images from "Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels"

    Article Title: Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-020-00880-y

    Multiplex bisulphite PCR amplicon sequencing of bisulphite-treated FFPET clinical DNA. a TapeStation gel showing 6 representative sequencing libraries from breast cancer FFPET-derived DNA samples are the correct sizes (~ 250 bp). EL = electronic ladder is shown. b Boxplot showing full coverage (top panel) across the 33 amplicons of the breast cancer panels from a sequencing run on our normal and tumour FFPET samples. Bottom panel shows the same data with a different y -axis scale to better show the difference between the lower coverage amplicons, with the dashed line indicating the cut-off (100 reads). c Line plots showing methylation data of 4 representative amplicons across 5 normal and 5 tumour clinical FFPET samples, demonstrating distinct separation between the methylation of normal and tumour samples
    Figure Legend Snippet: Multiplex bisulphite PCR amplicon sequencing of bisulphite-treated FFPET clinical DNA. a TapeStation gel showing 6 representative sequencing libraries from breast cancer FFPET-derived DNA samples are the correct sizes (~ 250 bp). EL = electronic ladder is shown. b Boxplot showing full coverage (top panel) across the 33 amplicons of the breast cancer panels from a sequencing run on our normal and tumour FFPET samples. Bottom panel shows the same data with a different y -axis scale to better show the difference between the lower coverage amplicons, with the dashed line indicating the cut-off (100 reads). c Line plots showing methylation data of 4 representative amplicons across 5 normal and 5 tumour clinical FFPET samples, demonstrating distinct separation between the methylation of normal and tumour samples

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Methylation

    20) Product Images from "Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels"

    Article Title: Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-020-00880-y

    Analysis of circulating cell-free tumour DNA using multiplex PCR bisulphite sequencing. a Gel showing successful MBPS libraries (~ 250 bp) of 24 breast cancer-derived circulating cfDNA samples (pre-PCR cleanup). L = ladder. b Boxplot showing full coverage (top panel) across the 33 amplicons of the breast cancer panels from a sequencing run on the 24 cfDNA tumour samples. Bottom panel shows the same data with a different y -axis scale to better show the difference between the lower coverage amplicons, with the dashed line indicating the cut-off (100 reads). c Boxplot of the methylation values detected using the MBPS assay across 24 cfDNA samples
    Figure Legend Snippet: Analysis of circulating cell-free tumour DNA using multiplex PCR bisulphite sequencing. a Gel showing successful MBPS libraries (~ 250 bp) of 24 breast cancer-derived circulating cfDNA samples (pre-PCR cleanup). L = ladder. b Boxplot showing full coverage (top panel) across the 33 amplicons of the breast cancer panels from a sequencing run on the 24 cfDNA tumour samples. Bottom panel shows the same data with a different y -axis scale to better show the difference between the lower coverage amplicons, with the dashed line indicating the cut-off (100 reads). c Boxplot of the methylation values detected using the MBPS assay across 24 cfDNA samples

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Bisulfite Sequencing, Derivative Assay, Sequencing, Methylation

    21) Product Images from "Centrifugation-free extraction of circulating nucleic acids using immiscible liquid under vacuum pressure"

    Article Title: Centrifugation-free extraction of circulating nucleic acids using immiscible liquid under vacuum pressure

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23766-9

    Comparison of extracted cfDNA concentration in qPCR assay. cfDNA concentration in a plasma sample was compared according to extraction methods, QIAamp and PIBEX. cfDNA amount in plasma sample was quantified by qPCR in four reference genes, TERT, RPPH1, GAPDH, and NAGK, in ( a ). The error bar represents standard deviation of triplex qPCR assay in a sample. The relative efficiency of cfDNA extraction in PIBEX was derived based on QIAamp in ( b ). The error bar is standard deviation of samples (n = 7).
    Figure Legend Snippet: Comparison of extracted cfDNA concentration in qPCR assay. cfDNA concentration in a plasma sample was compared according to extraction methods, QIAamp and PIBEX. cfDNA amount in plasma sample was quantified by qPCR in four reference genes, TERT, RPPH1, GAPDH, and NAGK, in ( a ). The error bar represents standard deviation of triplex qPCR assay in a sample. The relative efficiency of cfDNA extraction in PIBEX was derived based on QIAamp in ( b ). The error bar is standard deviation of samples (n = 7).

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay

    Related Articles

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    Article Title: Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)
    Article Snippet: .. The following kits were used: PME free-circulating DNA Extraction Kit protocol (Analytik Jena, Jena, Germany) for 2–5 mL extractions using lysis solution GS/Binding solution VL system and DSP Virus/Pathogen Midi Kit performed on QIAsymphony (Qiagen) and the QIAamp Circulating Nucleic Acid Kit (QIAGEN). .. QIAsymphony extractions were performed at the Qiagen applications lab (Hilden).

    Article Title: Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions
    Article Snippet: .. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). .. To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine.

    Isolation:

    Article Title: An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage
    Article Snippet: .. Cell-free DNA purification and quantification Circulating DNA was isolated from 1–5 mL plasma with the QIAamp Circulating Nucleic Acid Kit (Qiagen). .. The concentration of purified plasma DNA was determined by quantitative PCR (qPCR) using an 81 bp amplicon on chromosome 1 and a dilution series of intact male human genomic DNA (Promega) as a standard curve.

    Article Title: Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions
    Article Snippet: .. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). .. To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine.

    Sampling:

    Article Title: Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study
    Article Snippet: .. In summary, the optimal conditions identified in our study for the implementation of cfDNA analysis in routine diagnosis were (i) the sampling of patient blood in EDTA tubes and analysis within 24 hours, (ii) the extraction of cfDNA from at least 3 mL plasma using the QIAamp circulating nucleic acid kit from QIAgen to obtain a sufficient cfDNA input for the detection of 1,000 wild-type copies, (iii) the use of a highly sensitive and specific Droplet Digital PCR technology (Bio-Rad), (iv) the in-house design of primers/probes to amplify shorter amplicons and increase the sensitivity of the assay, and (v) the limitation of false-positive case by implementing for each targeted hot-spot, two distinct detection systems. .. Overall, our results argue in favor of the routine use of digital PCR, as pointed out within the AURA trial (NCT01802632, funded by Astra Zeneca) focusing on the detection of EGFR alterations in plasma by digital PCR (ddPCR and BEAMing), either as a complementary technique to tissue biopsies for the detection of mutations at diagnosis or as an alternative method for repeated analyses during tumor progression [ ].

    Digital PCR:

    Article Title: Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study
    Article Snippet: .. In summary, the optimal conditions identified in our study for the implementation of cfDNA analysis in routine diagnosis were (i) the sampling of patient blood in EDTA tubes and analysis within 24 hours, (ii) the extraction of cfDNA from at least 3 mL plasma using the QIAamp circulating nucleic acid kit from QIAgen to obtain a sufficient cfDNA input for the detection of 1,000 wild-type copies, (iii) the use of a highly sensitive and specific Droplet Digital PCR technology (Bio-Rad), (iv) the in-house design of primers/probes to amplify shorter amplicons and increase the sensitivity of the assay, and (v) the limitation of false-positive case by implementing for each targeted hot-spot, two distinct detection systems. .. Overall, our results argue in favor of the routine use of digital PCR, as pointed out within the AURA trial (NCT01802632, funded by Astra Zeneca) focusing on the detection of EGFR alterations in plasma by digital PCR (ddPCR and BEAMing), either as a complementary technique to tissue biopsies for the detection of mutations at diagnosis or as an alternative method for repeated analyses during tumor progression [ ].

    Article Title: Efficient treatment of a metastatic melanoma patient with a combination of BRAF and MEK inhibitors based on circulating tumor DNA analysis: a case report
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    DNA Purification:

    Article Title: An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage
    Article Snippet: .. Cell-free DNA purification and quantification Circulating DNA was isolated from 1–5 mL plasma with the QIAamp Circulating Nucleic Acid Kit (Qiagen). .. The concentration of purified plasma DNA was determined by quantitative PCR (qPCR) using an 81 bp amplicon on chromosome 1 and a dilution series of intact male human genomic DNA (Promega) as a standard curve.

    Article Title: Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions
    Article Snippet: .. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). .. To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine.

    Lysis:

    Article Title: Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)
    Article Snippet: .. The following kits were used: PME free-circulating DNA Extraction Kit protocol (Analytik Jena, Jena, Germany) for 2–5 mL extractions using lysis solution GS/Binding solution VL system and DSP Virus/Pathogen Midi Kit performed on QIAsymphony (Qiagen) and the QIAamp Circulating Nucleic Acid Kit (QIAGEN). .. QIAsymphony extractions were performed at the Qiagen applications lab (Hilden).

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    Efficiency of separate extraction of DNA and RNA when compared to total nucleic acid extraction using QIAamp® MinElute® Virus Spin kit. DNA and RNA were extracted separately and compared to the total nucleic acid extracted using the QIAamp® MinElute® Virus Spin kit, using qPCR. RNA extraction using the Qiagen RNeasy® Mini kit did not enhance the detection of the two single-stranded RNA viruses (FeLV and RSV), whereas the RNase A digestion added to the Wako DNA Extractor® kit greatly enriched for double-stranded nucleic acid (EBV and Reo3)

    Journal: NPJ Vaccines

    Article Title: Selection and evaluation of an efficient method for the recovery of viral nucleic acids from complex biologicals

    doi: 10.1038/s41541-018-0067-3

    Figure Lengend Snippet: Efficiency of separate extraction of DNA and RNA when compared to total nucleic acid extraction using QIAamp® MinElute® Virus Spin kit. DNA and RNA were extracted separately and compared to the total nucleic acid extracted using the QIAamp® MinElute® Virus Spin kit, using qPCR. RNA extraction using the Qiagen RNeasy® Mini kit did not enhance the detection of the two single-stranded RNA viruses (FeLV and RSV), whereas the RNase A digestion added to the Wako DNA Extractor® kit greatly enriched for double-stranded nucleic acid (EBV and Reo3)

    Article Snippet: The QIAGEN QIAamp® Circulating Nucleic Acid kit accepted a larger input volume (5 mL) with ease but recovery of single-stranded RNA viruses were approximately twofold less compared to baseline.

    Techniques: Real-time Polymerase Chain Reaction, RNA Extraction

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.21256

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

    Article Snippet: In summary, the optimal conditions identified in our study for the implementation of cfDNA analysis in routine diagnosis were (i) the sampling of patient blood in EDTA tubes and analysis within 24 hours, (ii) the extraction of cfDNA from at least 3 mL plasma using the QIAamp circulating nucleic acid kit from QIAgen to obtain a sufficient cfDNA input for the detection of 1,000 wild-type copies, (iii) the use of a highly sensitive and specific Droplet Digital PCR technology (Bio-Rad), (iv) the in-house design of primers/probes to amplify shorter amplicons and increase the sensitivity of the assay, and (v) the limitation of false-positive case by implementing for each targeted hot-spot, two distinct detection systems.

    Techniques: Concentration Assay

    Average cfDNA yield (ng/mL) in different solid tumor types studied cfDNA extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay cfDNA yield (ng/mL). n represent the number tested for each tumor type. * one sample excluded from graph due to high cfDNA concentration (769 ng/mL).

    Journal: Oncotarget

    Article Title: Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors

    doi: 10.18632/oncotarget.21982

    Figure Lengend Snippet: Average cfDNA yield (ng/mL) in different solid tumor types studied cfDNA extracted using the QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit dsDNA HS Assay cfDNA yield (ng/mL). n represent the number tested for each tumor type. * one sample excluded from graph due to high cfDNA concentration (769 ng/mL).

    Article Snippet: Plasma cfDNA was extracted from a 3-mL plasma sample using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions followed by elution in 50 μl.

    Techniques: Concentration Assay

    Detection of BRAF V600E mutations in the patient’s plasma. Plasma was collected every day when the patient was in the hospital (9 days), and at each clinical evaluation (after 4 and 8 weeks of treatment). DNA was extracted from plasma (2 mL) using the QIAamp circulating nucleic acid kit (Qiagen). BRAF V600E mutations were detected and quantified by digital PCR using the QuantStudio 3D system and a specific BRAF V600E probe (Thermo Fischer, Courtaboeuf, France)

    Journal: BMC Research Notes

    Article Title: Efficient treatment of a metastatic melanoma patient with a combination of BRAF and MEK inhibitors based on circulating tumor DNA analysis: a case report

    doi: 10.1186/s13104-017-2650-5

    Figure Lengend Snippet: Detection of BRAF V600E mutations in the patient’s plasma. Plasma was collected every day when the patient was in the hospital (9 days), and at each clinical evaluation (after 4 and 8 weeks of treatment). DNA was extracted from plasma (2 mL) using the QIAamp circulating nucleic acid kit (Qiagen). BRAF V600E mutations were detected and quantified by digital PCR using the QuantStudio 3D system and a specific BRAF V600E probe (Thermo Fischer, Courtaboeuf, France)

    Article Snippet: DNA was extracted from plasma samples using the QIAamp circulating nucleic acid kit (Qiagen, Courtaboeuf, France), and analyzed by digital PCR using the QuantStudio 3D System and specific probes (Thermo Fischer, Courtaboeuf, France).

    Techniques: Digital PCR