Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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DNA qualification workflow for next generation sequencing of histopathological samples.

Author(s): Simbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpeli G, Lawlor RT, Scarpa A

Publication: PLoS One, 2013, Vol. 8, Page e62692

PubMed ID: 23762227 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of quantification method, RNA interference, and DNA integrity on the quantification of DNA from FFPE and frozen specimens. DNA was extracted from 6 frozen tissues (five pancreas and one spleen) stored at -80°C using QIAmp AllPrep DNA/RNA mini kit and from 6 FFPE tissues (four pancreas, one duodenum and one liver) using the QIAmp DNA FFPE tissue kit.

   Summary of Findings:

   With the exception of one frozen specimen, measured levels of DNA were higher when quantified using NanoDrop then when quantified using Qubit. Electrophoresis and real-time PCR showed the DNA was intact, and QuBit quantification was accurate, but that NanoDrop overestimated the DNA concentration. Further, dilution of the DNA did not increase the accuracy of quantification. For high molecular weight DNA, the performance of Qubit and NanoDrop were comparable, but when the DNA was degraded, such as in the FFPE specimens, Qubit performed better. When RNA was added to DNA, it interfered with determination of DNA concentration by NanoDrop, but Qubit was only affected when the sample had 4-fold more RNA than DNA. Importantly, when NanoDrop was used to quantify DNA for construction of a next-generation sequencing library from FFPE specimens, no amplification was observed, but when DNA was quantified using Qubit, the library was adequate for sequencing in 2 of 3 specimens with the single failure due to organic contamination.

   Biospecimens

   • Tissue - Liver
   • Tissue - Small Bowel
   • Tissue - Spleen
   • Tissue - Pancreas

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis
   DNA	Real-time qPCR
   DNA	Spectrophotometry
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen
   Spectrophotometry Specific	Technology platform	Qubit NanoDrop Electrophoresis Real-time PCR
   Spectrophotometry Specific	Template/input amount	Diluted with RNA Diluted 1:1 Diluted 1:5 Diluted 1:10

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