Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

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

Conclusion of Paper

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

   • Tissue - Liver

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Hepatitis
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Spectrophotometry
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	PCR
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(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/input amount	Not diluted Diluted in distilled water Diluted in TE
   Real-time qPCR Specific	Template/input amount	Diluted in TE Diluted in distilled water Not diluted
   Spectrophotometry Specific	Template/input 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

   View more

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

   • Tissue - Liver

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Hepatitis
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Electrophoresis
   DNA	PCR
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Fluorometry Specific	Template/input 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

   View more