Leah R. Turner, Victor Sementchenko and Luke Sherlin
NuGEN Technologies, Inc. San Carlos, CA 94070.
Gene expression analysis of clinical samples has long presented challenges to researchers based on sample availability, amount, and integrity. These challenges are most significant for small and typically degraded formalin-fixed, paraffin-embedded (FFPE) samples widely used in cancer research and clinical diagnostics. The whole transcriptome approach, used in NuGEN’s Ovation RNA-Seq FFPE System, enables amplification and sample preparation of FFPE total RNA through a simple, robust, and easily automatable solution specifically for accurate and robust analysis on next generation sequencing (NGS) platforms. In this study, RNA isolated from FFPE and Fresh Frozen (FF) matched Colon Tumor and Normal Adjacent Tissue (NAT) pairs was amplified. The amplified products were used to prepare Next Generation Sequencing libraries with the NuGEN Encore NGS Multiplex System I and sequenced using the Illumina GAIIx platform.
Principal Component Analysis (PCA) showed that samples completely segregated by disease state, demonstrating that the amplification of FFPE RNA maintains the integrity of the biological data. ANOVA was performed to identify differentially expressed genes between NAT and Colon Tumor pairs. Fold changes as low as 1.3-fold were detected in the FFPE samples at a P-value cut off of 0.001, when 4 Tumor/NAT pairs were analyzed. Concordance of differential expression between quad set samples (matched FFPE and FF) was demonstrated, with concordance in 97% of the genes identified as differentially expressed at a P-value cut off of 0.01. Pathway analysis demonstrated that the biology suggested by the PCA is, indeed, detectable in FFPE samples. The NuGEN Ovation RNA-Seq FFPE System enables NGS-based expression profiling and allows the vast archives of valuable FFPE samples to be accessed for clinical research projects and makes routine clinical analysis of FFPE samples a possibility.
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