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Lookup NU author(s): Lynsey Rogerson, Dr Steven Darby, Dr Talal Jabbar, Dr Marie Mathers, Professor Hing Leung, Professor Craig Robson, Dr Kanagasabai Sahadevan, Kieran O'Toole, Vincent Gnanapragasam
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OBJECTIVE: To investigate the feasibility of transcript profiling in diagnostic formalin-fixed and paraffin-embedded (FFPE) biopsies for prostate cancer. MATERIALS AND METHODS: 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. RESULTS: 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 application in detecting differences in EZH2 and Id-1 expression between malignant and benign epithelium. The results confirmed that our technique was capable of quantifying significant differences in expression between malignant and benign epithelium consistent with the reported trends. CONCLUSION: This study reports the use of standard FFPE needle biopsies for transcript profiling and supports the concept of molecular prognostic studies in tissue acquired at diagnosis in prostate cancer. © 2008 The Authors.
Author(s): Rogerson LC, Darby S, Jabbar TA, Mathers ME, Leung HY, Robson CN, Sahadevan K, O'Toole KT, Gnanapragasam VJ
Publication type: Article
Publication status: Published
Journal: BJU International
Year: 2008
Volume: 102
Issue: 3
Pages: 364-370
ISSN (print): 1464-4096
ISSN (electronic): 1464-410X
Publisher: Wiley-Blackwell Publishing Ltd.
URL: http://dx.doi.org/10.1111/j.1464-410X.2008.07627.x
DOI: 10.1111/j.1464-410X.2008.07627.x
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