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[1586] Rapid Quantitative Measurment of Gene Expression in Formalin-Fixed Tissues and LCM Samples without RNA Purification Using the QuantiGene Branched-Chain DNA Assay

A Allen, D McLerran, J Davies, B Vessella, G McMaster, A Kristal, BS Knudsen. Fred Hutchinson Cancer Center, Seattle, WA; U Washington, Seattle, WA; Panomics, Fremont, CA

Background: The difficulty in measuring RNA concentrations in FFPE tissues arises from extensive fragmentation and crosslinking of RNA after formalin fixation. The QuantiGene (QG) assay does not require RNA purification and relies on cooperative hybridization: its probe design and non-enzymatic RNA capture and detection overcome the adverse effects of formalin fixation. The QG technology uses an ELISA-type format and is ideally suited for simple, rapid and high-throughput sample preparation and measurement of gene expression panels in FFPE tissue homogenates.
Design: Duplicate samples from 10 different xenografts were snap frozen or fixed in formalin. RNA was measured either after isolation (pRNA) or after solubilization in a tissue homogenization buffer (thRNA). Probe sets for capture of formalin-fixed (FF) RNA were specifically designed for short RNA fragments. To validate the QG assay, we measured the expression of six genes in 20 samples in parallel by qPCR, Agilent 2100 bioanalyzer and QG assay and obtained correlation coefficients directly from the variance components model. To evaluate the reliability of the QG assay, we determined the intraclass correlation coefficient (ICC). Laser capture microdissection (LCM) was performed with the Veritas instrument.
Results: Measurements using the QG assay for RNA preparations from FF tissues were highly reproducible, with ICCs of six genes ranging between 0.84 and 0.97. The correlation coefficients for measurements of gene expression by QG assay in FF and frozen tissues compared to qPCR measurements of frozen tissues were between 0.53 and 0.93 and between 0.22 and 0.93, respectively. In contrast, the correlation coefficients of qPCR measurements ranged between -0.33 and 0.72. The sensitivity of the QG assay in FF tissues was comparable to qPCR for pRNA, however it was significantly increased for tissue homogenates. The QG assay accurately measures 28S and18S RNA and 18S DNA in fewer than 5000 cells. Measuring a gene expression profile in LCM tissues that distinguishes cancer from normal epithelium provided the expected increases of gene expression in laser captured prostate cancer glands.
Conclusions: The QuantiGene assay permits measurements of RNA in tissue homogenates without RNA isolation and provides a rapid workflow and better reproducibility, accuracy and sensitivity than PCR-based methods.
Category: Techniques

Monday, March 26, 2007 1:30 PM

Platform Session: Section G, Monday Afternoon