Journal of Medical Diagnostic Methods
Open Access

Abstract

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

Kiran T Malhotra, Uday Gulati, Bonnie Balzer and Helen Y Wu

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