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...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4777359?pdf=render |
| _version_ | 1818049807727460352 |
|---|---|
| author | Yuki Nakayama Hiromi Yamaguchi Naoki Einaga Mariko Esumi |
| author_facet | Yuki Nakayama Hiromi Yamaguchi Naoki Einaga Mariko Esumi |
| author_sort | Yuki Nakayama |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-10T10:43:27Z |
| format | Article |
| id | doaj.art-2811c4237c4c41feba4a727fd22f0595 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T10:43:27Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-2811c4237c4c41feba4a727fd22f05952022-12-22T01:52:14ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01113e015052810.1371/journal.pone.0150528Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.Yuki NakayamaHiromi YamaguchiNaoki EinagaMariko EsumiThe 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.http://europepmc.org/articles/PMC4777359?pdf=render |
| spellingShingle | Yuki Nakayama Hiromi Yamaguchi Naoki Einaga Mariko Esumi Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC4777359?pdf=render |
| work_keys_str_mv | AT yukinakayama pitfallsofdnaquantificationusingdnabindingfluorescentdyesandsuggestedsolutions AT hiromiyamaguchi pitfallsofdnaquantificationusingdnabindingfluorescentdyesandsuggestedsolutions AT naokieinaga pitfallsofdnaquantificationusingdnabindingfluorescentdyesandsuggestedsolutions AT marikoesumi pitfallsofdnaquantificationusingdnabindingfluorescentdyesandsuggestedsolutions |