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A rapid and simple method for the detection of prostate-specific antigen mRNA in archival tissue specimens using a
R A Edelstein1, R J Krane, R K Babayan
1Department of Urology, Boston University School of Medicine, Massachusetts, USA.
Objectives:
Polymerase chain reaction (PCR) amplification of DNA from archival, fixed tissue sources is now performed routinely. In this report, we describe a reproducible technique for mRNA amplification from archival tissues.
Methods:
Archival, fixed tissue was treated with a modification of a rapid, acid guanidinium technique. The RNA yielded was reverse transcribed and subjected to 40 cycles of two-step PCR, using a panel of primers designed to encompass relatively short (less than 250 bp) cDNA fragments. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining.
Results:
Using fixed, paraffin-embedded specimens of human prostate, we have demonstrated a reproducible pattern of reverse transcription (RT)-PCR products using several different primer sets. Our data suggest that RNA degradation proceeds to fragments approximately 250 bp or shorter in length but that many of these fragments survive intact over extended periods of time and are of suitable quality to serve as a template for RT and subsequent PCR amplification.
Conclusions:
We describe a technique that will allow the retrospective analysis of archival tissues for gene expression. These methods are generally applicable to a wide variety of systems. Such a method will make it possible to perform retrospective studies of gene expression in the archival tissues of patients whose eventual clinical course is already known, greatly shortening the time needed for genetic outcome studies in slowly growing tumors, such as prostate cancer.