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Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions
The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most ac...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777359/ https://www.ncbi.nlm.nih.gov/pubmed/26937682 http://dx.doi.org/10.1371/journal.pone.0150528 |
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author | Nakayama, Yuki Yamaguchi, Hiromi Einaga, Naoki Esumi, Mariko |
author_facet | Nakayama, Yuki Yamaguchi, Hiromi Einaga, Naoki Esumi, Mariko |
author_sort | Nakayama, Yuki |
collection | PubMed |
description | The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR. |
format | Online Article Text |
id | pubmed-4777359 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-47773592016-03-10 Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions Nakayama, Yuki Yamaguchi, Hiromi Einaga, Naoki Esumi, Mariko PLoS One Research Article The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR. Public Library of Science 2016-03-03 /pmc/articles/PMC4777359/ /pubmed/26937682 http://dx.doi.org/10.1371/journal.pone.0150528 Text en © 2016 Nakayama et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Nakayama, Yuki Yamaguchi, Hiromi Einaga, Naoki Esumi, Mariko Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions |
title | Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions |
title_full | Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions |
title_fullStr | Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions |
title_full_unstemmed | Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions |
title_short | Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions |
title_sort | pitfalls of dna quantification using dna-binding fluorescent dyes and suggested solutions |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777359/ https://www.ncbi.nlm.nih.gov/pubmed/26937682 http://dx.doi.org/10.1371/journal.pone.0150528 |
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