Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5 ' nuclease quantitative reverse transcription-polymerasechain reaction

Analysis of gene expression and correlation with clinical parameters has th e potential to become an important factor in therapeutic decision making. T he ability to analyze gene expression in archived tissues, for which clinic al followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accu rately reflect expression before fixation. In the present study we have opt imized the RNA isolation and reverse transcription steps for quantitative r everse transcription-polymerase chain reaction (RT-PCR) on archival materia l. Using tissue taken directly from the operating room, mRNAs with half-liv es from 10 minutes to >8 hours were isolated and reverse transcribed. Subse quent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fi xation times (time from excision to fixation) of up to 12 hours, Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.