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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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Significant discrepancies in DNA quantification by NanoDrop and Qubit.A total of 100 ng of DNA based on NanoDrop (N, black bars) or Qubit (Q, grey bars) measurements was analyzed by electrophoresis on 0.8% agarose gel. Sample ID is indicated at the bottom. Lane L contains 200 ng of DNA as the reference for normalization. Densitometric analysis (bar chart) was performed by ImageJ software [20]. It is clear from the electrophoretic bands and their densitometric charts that NanoDrop overestimates DNA concentration.
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pone-0062692-g002: Significant discrepancies in DNA quantification by NanoDrop and Qubit.A total of 100 ng of DNA based on NanoDrop (N, black bars) or Qubit (Q, grey bars) measurements was analyzed by electrophoresis on 0.8% agarose gel. Sample ID is indicated at the bottom. Lane L contains 200 ng of DNA as the reference for normalization. Densitometric analysis (bar chart) was performed by ImageJ software [20]. It is clear from the electrophoretic bands and their densitometric charts that NanoDrop overestimates DNA concentration.

Mentions: NanoDrop concentration values were higher than Qubit for all samples but sample A (Table 2). To confirm this observation and assay DNA quality, 100 ng of DNA based on either NanoDrop or Qubit measurements were loaded side by side on a 0.8% agarose gel (Figure 2). All DNAs showed satisfactory DNA integrity without evident smears, with the exception of sample FF46 (not shown). Densitometric analysis confirmed that NanoDrop overestimated the DNA content of most samples (Figure 2), while Qubit measures were consistent with the densitometric evaluation of agarose gel bands of integer DNA.


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)

Significant discrepancies in DNA quantification by NanoDrop and Qubit.A total of 100 ng of DNA based on NanoDrop (N, black bars) or Qubit (Q, grey bars) measurements was analyzed by electrophoresis on 0.8% agarose gel. Sample ID is indicated at the bottom. Lane L contains 200 ng of DNA as the reference for normalization. Densitometric analysis (bar chart) was performed by ImageJ software [20]. It is clear from the electrophoretic bands and their densitometric charts that NanoDrop overestimates DNA concentration.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062692-g002: Significant discrepancies in DNA quantification by NanoDrop and Qubit.A total of 100 ng of DNA based on NanoDrop (N, black bars) or Qubit (Q, grey bars) measurements was analyzed by electrophoresis on 0.8% agarose gel. Sample ID is indicated at the bottom. Lane L contains 200 ng of DNA as the reference for normalization. Densitometric analysis (bar chart) was performed by ImageJ software [20]. It is clear from the electrophoretic bands and their densitometric charts that NanoDrop overestimates DNA concentration.
Mentions: NanoDrop concentration values were higher than Qubit for all samples but sample A (Table 2). To confirm this observation and assay DNA quality, 100 ng of DNA based on either NanoDrop or Qubit measurements were loaded side by side on a 0.8% agarose gel (Figure 2). All DNAs showed satisfactory DNA integrity without evident smears, with the exception of sample FF46 (not shown). Densitometric analysis confirmed that NanoDrop overestimated the DNA content of most samples (Figure 2), while Qubit measures were consistent with the densitometric evaluation of agarose gel bands of integer DNA.

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