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Diagnostic Technologies and Molecular and Cellular Profiling: Biomarker Assay Development, Validation, and Qualification

Multiplex qRT-PCR gene expression assay on the COBAS^® TaqMan 48 Analyzer using RNA extracted from formalin-fixed paraffin embedded tissue

Roche Molecular Systems, Pleasanton, CA and Response Genetics, Inc., Los Angeles, CA

Abstract

A11

Roche Molecular Systems has partnered with external pharmaceutical companies to develop clinical diagnostic assays that may provide patients with improved therapies. Companion diagnostics utilizing valid biomarkers coupled with certain drugs may be used for the purpose of patient stratification. In order to facilitate the development of companion diagnostic gene expression tests, a total RNA extraction method has been optimized for formalin-fixed paraffin embedded (FFPE) tissue and a qRT-PCR gene expression assay has been evaluated on the COBAS® TaqMan 48 Analyzer.Archived formalin-fixed paraffin-embedded tissue samples are an invaluable resource for gene-expression profiling in cancer patients. FFPE tissue samples are often readily available and can be associated with clinical outcome. However, extraction of quality RNA from FFPE tissue has proven to be problematic due to cross-linking of proteins and nucleotides during the formalin fixation process. We have evaluated several FFPE tissue RNA extraction kits including the High Pure RNA Paraffin Kit (Roche Applied Science) and the RNeasy® FFPE Kit (Qiagen). Based on the initial evaluation, the RNeasy® FFPE Kit was selected for further studies due to shorter processing time of 90 minutes compared to 24 hours for the High Pure RNA Paraffin Kit. Both kits had comparable performance based on amount of RNA extracted and the ability to amplify this RNA.Analytical performance of a multiplex qRT-PCR gene expression assay using COBAS® TaqMan 48 Analyzer for real-time single-tube reverse transcription, amplification and detection was evaluated. Linearity studies demonstrated R² values were >0.97 for the all four genes of the multiplex reaction when testing 2-fold serial dilutions (20 -0.156 ng) of Universal Human Reference RNA (Stratagene). Furthermore, assay linearity and precision using micro-dissected clinical samples extracted with the RNeasy® FFPE Kit will be presented. The current results indicate that we have a fast, non-organic, IVD compatible RNA extraction method for FFPE tissue that can provide quality RNA for multiplex real time qRT-PCR assays on the COBAS® TaqMan 48 Analyzer.