alexa Comparison of DNA Extraction Methods from Formalin-Fixe
ISSN: 2168-9784

Journal of Medical Diagnostic Methods
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Research Article

Comparison of DNA Extraction Methods from Formalin-Fixed, Paraffin- Embedded Tissue and their Impact on Real-Time PCR-Based Mutation Assays

Kiran T Malhotra1*, Uday Gulati2, Bonnie Balzer3 and Helen Y Wu1
1Genomics and Oncology Research Department, Roche Molecular Systems, Inc., Pleasanton, California, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
3Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California, USA
Corresponding Author : Kiran T Malhotra
Global Development RMD
Roche Diagnostics International Ltd
Forrenstrasse 2, 6343 Rotkreuz, Switzerland
Tel: + 41-41-798-5642
E-mail: [email protected]
Received July 25, 2012; Accepted August 21, 2012; Published August 24, 2012
Citation: Malhotra KT, Gulati U, Balzer B, Wu HY (2012) Comparison of DNA Extraction Methods from Formalin-Fixed, Paraffin-Embedded Tissue and their Impact on Real-Time PCR-Based Mutation Assays. J Med Diagn Meth 1:107. doi:10.4172/2168-9784.1000107
Copyright: © 2012 Malhotra KT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 

Abstract

In this new era of personalized cancer care, molecular testing for somatic mutations plays an increasing role in treatment decisions for many targeted cancer therapeutics. Formalin-fixed paraffin-embedded tissue (FFPET) specimens remain the typical source of nucleic acid for such testing. Because formalin fixation can damage nucleic acids and many tumor specimens are small, obtaining a sufficient quantity of high-quality DNA for mutation testing can be challenging. Given these pre-analytic variables, the availability of a standardized and well-validated method to isolate DNA from such specimen types is critical. We compared two widely available commercial kits for DNA isolation (cobas DNA Sample Preparation Kit from Roche Molecular Systems, and QIAamp DNA FFPE Tissue Kit from Qiagen) using 120 FFPET specimens from a range of tumor types (melanoma, thyroid, colorectal, lung, breast, and ovarian cancer) and examined the effects of the different DNA isolation methods on the subsequent performance of real-time PCR-based assays for BRAF, KRAS and EGFR mutations. Although the two methods gave comparable nucleic acid quantities, the cobas method co-purified significantly less RNA (p<0.001) as determined by comparing DNA yields before and after RNase treatment. The presence of RNA in the extracted DNA was associated with delayed threshold cycle (Ct) in real-time PCR-based mutation tests. The cobas method consistently yielded a sufficient quantity of purified DNA across range of tumor types and specimen sizes for real-time PCR mutation detection tests without requiring an additional step of RNase treatment

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