Application of transcript profiling in formalin-fixed paraffin-embedded diagnostic prostate cancer needle biopsies.
Abstract
To investigate the feasibility of transcript profiling in diagnostic formalin-fixed and paraffin-embedded (FFPE) biopsies for prostate cancer. Laser-capture microdissection (LCM) was used to microdissect glandular epithelium as well as stromal tissue in archival prostate needle biopsies. Optimized RNA extraction, reverse transcription and real-time PCR (QPCR) protocols were used to detect transcript expression. RNA degradation effects were assessed using hydrolysed cell line RNA and matched xenograft FFPE and frozen tumours. LCM and RNA extraction was achieved in all biopsies from a pilot cohort of five patients. cDNA produced was successfully used to detect expression of glyceraldehyde-3-phosphate dehydrogenase, RPL13, prostate-specific antigen, vimentin, inhibitor of differentiation/DNA binding 1 (Id-1) and polycomb group protein enhancer of zeste homolog 2 (EZH2) transcripts. In the cell line and xenograft models, we investigated the effect of RNA degradation on transcript quantification by QPCR. In both models normalization of transcript quantity with a housekeeping gene resulted in restored expression in all degraded samples to within a 50% difference of control samples. Using an extended cohort of 29 biopsies, we tested a...Continue Reading
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