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Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions
Yuki Nakayama1, Hiromi Yamaguchi1, Naoki Einaga1
1Department of Pathology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan.
Plos One
|March 4, 2016
Summary
The Qubit fluorometer is not always the most accurate DNA quantification method. For formalin-fixed, paraffin-embedded DNA, quantitative PCR (qPCR) better assesses fragmentation than Qubit.
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- Accurate DNA quantification is crucial for downstream applications like next-generation sequencing.
- The Qubit fluorometer measures double-stranded DNA (dsDNA) via fluorescence, often used for quality control.
- Comparing different DNA quantification methods is essential for selecting the most suitable technique for specific sample types.
Purpose of the Study:
- To compare the accuracy and suitability of NanoDrop, Qubit, and quantitative PCR (qPCR) for DNA quantification.
- To evaluate these methods across different DNA extraction methods and preservation states: fresh frozen, FFPE, and Trizol-extracted DNA.
- To determine the optimal method for assessing DNA quality, particularly for FFPE samples.
Main Methods:
- Three DNA samples were analyzed: fresh frozen (Frozen-DNA), formalin-fixed, paraffin-embedded (FFPE-DNA), and Trizol-extracted (Trizol-DNA).
- DNA samples were serially diluted and quantified using NanoDrop (UV absorbance), Qubit (fluorescence), and qPCR (target gene abundance).
- Electrophoresis and single-stranded DNA (ssDNA) quantification were used to assess DNA integrity and composition.
Main Results:
- Qubit measurements for Frozen-DNA were not proportional to dilution without sufficient salt concentration (>1 mM NaCl).
- For FFPE-DNA, Qubit values were proportional but lower than NanoDrop; qPCR and electrophoresis indicated Qubit underestimated fragmentation.
- Trizol-DNA, primarily ssDNA, showed proportional Qubit readings but qPCR values were higher than NanoDrop, suggesting Qubit's limitations with ssDNA.
Conclusions:
- Qubit accuracy for DNA quantification is dependent on salt concentration and DNA integrity.
- Quantitative PCR (qPCR) is superior to Qubit for assessing DNA fragmentation in FFPE samples.
- Qubit is not universally the most accurate method for quantifying DNA, especially when dealing with ssDNA or fragmented DNA for PCR applications.
