NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

Author(s): Nakayama Y, Yamaguchi H, Einaga N, Esumi M

Publication: PLoS One, 2016, Vol. 11, Page e0150528

PubMed ID: 26937682 PubMed Review Paper? No

Purpose of Paper

This paper compared results obtained with different DNA quantification methods (NanoDrop, Qubit or real-time PCR) and diluents (water versus TE)  using frozen specimens, formalin-fixed paraffin-embedded (FFPE) specimens and samples post-RNA extraction with Trizol. The effects of diluents and prior extraction using Trizol on the percentage of DNA that was single- versus double-stranded were also investigated. 

Conclusion of Paper

DNA from frozen specimens or samples post-RNA extraction by Trizol were best quantified by qPCR or NanoDrop. Although both Qubit and PCR were suitable for quantification of DNA from FFPE specimens, qPCR alone accurately determined the degree of fragmentation. Dilution of DNA in water rather than TE or extraction with Trizol denatured the DNA such that almost all of the DNA was single stranded and thus couldn’t be quantified by Qubit or properly visualized using intercalating dyes on a TapeStation or gel.

Studies

  1. Study Purpose

    This study compared results obtained with different  DNA quantification methods (NanoDrop, Qubit or real-time PCR) and diluents (water versus TE) using frozen specimens, FFPE specimens and specimens previously subjected to RNA extraction using Trizol. Non-tumorous liver specimens from 6 patients with liver metastasis of colorectal carcinoma were stored at -80˚C until extraction using proteinase K and phenol-chloroform. Case-matched specimens from 3 of these patients were fixed in formalin for 2-4 days and extracted using a modified version of the RecoverAll Total Nucleic Acid Isolation Kit for FFPE. DNA was also extracted using trizol from 7 normal liver specimens from patients with HCV-positive hepatocellular carcinoma after RNA extraction of the same sample. DNA was diluted in water, TE or NaCl solution prior to quantification by spectrophotometer (Nanodrop), Qubit dsDNA BR (broad range, 2 to 1000 ng) and HS (high sensitivity, 0.2 to 100 ng) assays, and amplification of a 157 bp amplicon of GAPDH.  DNA integrity was assessed by PCR of 5 different amplicons of the Golgi membrane protein 1, TapeStation and gel electrophoresis.

    Summary of Findings:

    DNA concentrations were proportional to sample dilution when DNA extracted from frozen specimens or samples post-RNA extraction by Trizol was quantified by NanoDrop or real-time PCR. DNA concentrations were comparable between real-time PCR and Nanodrop for frozen specimens, but levels were approximately 50% higher by real-time PCR than NanoDrop for samples post-RNA extraction. In contrast, DNA levels for frozen specimens diluted by 8-fold or more with distilled water were lower than expected with Qubit, as were DNA levels in samples post-RNA extraction regardless of the diluent. Interestingly, DNA concentration was only slightly lower than when quantified by Qubit than NanoDrop (72% versus 21% when diluted in water). Using Rat DNA it was shown that quantification by Qubit required 1 mM or more sodium chloride present in the diluent, as addition of salt after dilution in distilled water failed to reverse the decline in DNA concentration.

     

    For FFPE specimens, real-time PCR and Qubit methods produced comparable DNA concentrations that were proportional to the distilled water dilution factor, but were much lower than concentrations determined by NanoDrop (p=0.01 and p=0.003, respectively), which was attributed to NanoDrop's quantification of degraded DNA. Interestingly, the ratio of Qubit to NanoDrop DNA concentration was consistent among specimens, but the same was not true for the ratio of real-time PCR to NanoDrop concentration, indicating differences between the two methods. Amplification efficiency and maximum PCR product sizes depended on the degradation of the specimen; thus, real-time PCR concentrations varied between specimens. Consequently, the authors conclude real-time PCR was the most reliable method for DNA quantification of FFPE specimens as it was the only method that quantified degradation.

    Biospecimens
    Preservative Types
    • Frozen
    • Formalin
    Diagnoses:
    • Neoplastic - Carcinoma
    • Hepatitis
    • Neoplastic - Normal Adjacent
    Platform:
    AnalyteTechnology Platform
    DNA Fluorometry
    DNA Spectrophotometry
    DNA Automated electrophoresis/Bioanalyzer
    DNA PCR
    DNA Real-time qPCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    Frozen
    Spectrophotometry Specific Technology platform Qubit
    NanoDrop
    Real-time PCR
    PCR Specific Technology platform TapeStation
    Electrophoresis
    Fluorometry Specific Template amount Not diluted
    Diluted in distilled water
    Diluted in TE
    Real-time qPCR Specific Template amount Diluted in TE
    Diluted in distilled water
    Not diluted
    Spectrophotometry Specific Template amount Not diluted
    Diluted in distilled water
    Diluted in TE
    PCR Specific Length of gene fragment 317 bp
    499 bp
    741 bp
    1357 bp
    2995 bp
    Analyte Extraction and Purification Analyte isolation method RNA extracted first
    RNA not extracted
  2. Study Purpose

    This study investigated the effects of diluent (water versus TE), and prior RNA extraction using Trizol on the on the percentage of DNA that is single- versus double-stranded.  Non-tumorous liver specimens from 6 patients with liver metastasis of colorectal carcinoma were stored at -80˚C until DNA extraction using proteinase K and phenol-chloroform. DNA was also extracted using trizol from 7 normal liver specimens from patients with HCV-positive hepatocellular carcinoma after RNA extraction. DNA was diluted in water, TE or NaCl solution prior to quantification by spectrophotometer (Nanodrop), Qubit dsDNA BR (broad range, 2 to 1000 ng) and HS (high sensitivity, 0.2 to 100 ng) assays, and amplification of a 157 bp amplicon of GAPDH. DNA integrity was assessed on a Tapestation and single stranded DNA was quantified using a combination of the Qubit dsDNA HS Assay Kit and the Qubit ssDNA assays.

    Summary of Findings:

    Double-stranded DNA comprised 92.3% of DNA extracted from frozen specimens when diluted with TE, but only 3.5% of the DNA when diluted with water. When DNA was extracted from samples post-RNA extraction with Trizol 94.5-97.% of the DNA was single-stranded before dilution occurred. Further confirming that prior Trizol extraction resulted in single-stranded DNA, amplification of all 5 PCR products including that of a 2995 bp fragment was possible in post-RNA extraction samples although the largest fragment was only visible as a weak smear by TapeStation and gel electrophoresis. Declines in quantifiable DNA after dilution in water or prior RNA extraction with Trizol are also consistent with a conformational DNA change from double-stranded to single-stranded.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    • Hepatitis
    • Neoplastic - Normal Adjacent
    Platform:
    AnalyteTechnology Platform
    DNA Fluorometry
    DNA Electrophoresis
    DNA PCR
    DNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Fluorometry Specific Template amount Diluted in water
    Diluted in TE
    PCR Specific Length of gene fragment 317 bp
    499 bp
    741 bp
    1357 bp
    2995 bp
    PCR Specific Technology platform TapeStation
    Electrophoresis
    Analyte Extraction and Purification Analyte isolation method RNA extracted first
    RNA not extracted

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