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DNA qualification workflow for next generation sequencing of histopathological samples.

Simbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpeli G, Lawlor RT, Scarpa A - PLoS ONE (2013)

Bottom Line: Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results.NanoDrop UV-spectrum verified contamination of the unsuccessful sample.In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

View Article: PubMed Central - PubMed

Affiliation: ARC-NET Research Centre, University of Verona, Verona, Italy.

ABSTRACT
Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

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Influence of RNA contamination on DNA quantification.DNA quantifications (n = 5) by NanoDrop and Qubit in the presence of RNA contamination. A DNA sample with a concentration of 38 ng/µl was mixed with different volumes of total RNA at 33 ng/µl extracted from the same tissue sample to obtain the indicated ratios; bars and brackets indicate mean and 95% confidence interval; asterisks show measurements significantly different from pure DNA (* p<0.05; ** p<0.001; Dunnett's post-hoc test). NanoDrop measurements were heavily influenced by the presence of RNA contamination (ANOVA p<0.0001), whereas Qubit values were less affected (ANOVA p<0.01). Black bars  =  NanoDrop; gray bars  =  Qubit.
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pone-0062692-g004: Influence of RNA contamination on DNA quantification.DNA quantifications (n = 5) by NanoDrop and Qubit in the presence of RNA contamination. A DNA sample with a concentration of 38 ng/µl was mixed with different volumes of total RNA at 33 ng/µl extracted from the same tissue sample to obtain the indicated ratios; bars and brackets indicate mean and 95% confidence interval; asterisks show measurements significantly different from pure DNA (* p<0.05; ** p<0.001; Dunnett's post-hoc test). NanoDrop measurements were heavily influenced by the presence of RNA contamination (ANOVA p<0.0001), whereas Qubit values were less affected (ANOVA p<0.01). Black bars  =  NanoDrop; gray bars  =  Qubit.

Mentions: To estimate the influence of RNA contamination on DNA quantification, genomic DNA (sample 148) at a concentration of 38 ng/µl was mixed with different volumes of total RNA at a concentration of 33 ng/µl, extracted from the same tissue sample, to obtain DNA/RNA ratios of 1∶1, 1∶2, 1∶4, 1∶8, and 1∶16 (Figure 4).


DNA qualification workflow for next generation sequencing of histopathological samples.

Simbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpeli G, Lawlor RT, Scarpa A - PLoS ONE (2013)

Influence of RNA contamination on DNA quantification.DNA quantifications (n = 5) by NanoDrop and Qubit in the presence of RNA contamination. A DNA sample with a concentration of 38 ng/µl was mixed with different volumes of total RNA at 33 ng/µl extracted from the same tissue sample to obtain the indicated ratios; bars and brackets indicate mean and 95% confidence interval; asterisks show measurements significantly different from pure DNA (* p<0.05; ** p<0.001; Dunnett's post-hoc test). NanoDrop measurements were heavily influenced by the presence of RNA contamination (ANOVA p<0.0001), whereas Qubit values were less affected (ANOVA p<0.01). Black bars  =  NanoDrop; gray bars  =  Qubit.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675123&req=5

pone-0062692-g004: Influence of RNA contamination on DNA quantification.DNA quantifications (n = 5) by NanoDrop and Qubit in the presence of RNA contamination. A DNA sample with a concentration of 38 ng/µl was mixed with different volumes of total RNA at 33 ng/µl extracted from the same tissue sample to obtain the indicated ratios; bars and brackets indicate mean and 95% confidence interval; asterisks show measurements significantly different from pure DNA (* p<0.05; ** p<0.001; Dunnett's post-hoc test). NanoDrop measurements were heavily influenced by the presence of RNA contamination (ANOVA p<0.0001), whereas Qubit values were less affected (ANOVA p<0.01). Black bars  =  NanoDrop; gray bars  =  Qubit.
Mentions: To estimate the influence of RNA contamination on DNA quantification, genomic DNA (sample 148) at a concentration of 38 ng/µl was mixed with different volumes of total RNA at a concentration of 33 ng/µl, extracted from the same tissue sample, to obtain DNA/RNA ratios of 1∶1, 1∶2, 1∶4, 1∶8, and 1∶16 (Figure 4).

Bottom Line: Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results.NanoDrop UV-spectrum verified contamination of the unsuccessful sample.In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

View Article: PubMed Central - PubMed

Affiliation: ARC-NET Research Centre, University of Verona, Verona, Italy.

ABSTRACT
Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Show MeSH