A rapid RT-PCR method for detection of intact RNA in formalin-fixed paraffin-embedded tissues.

The molecular analysis of formalin-fixed and paraffin-embedded tissues has application in both investigative studies and clinical practice (1). It is now possible to analyze gene expression in formalin-fixed and paraffin-embedded tissues by in situ reverse transcriptase-polymerase chain reaction (in situ RT-PCR) (2). Degradation of RNA, however, can occur at various stages of tissue preparation, fixation and paraffin-embedding. To date, a rapid technique designed to determine RNA quality in formalinfixed and paraffin-embedded tissues is lacking. The most common method for monitoring RNA involves tissue extraction using guanidinium thiocyanate or acid guanidinium thiocyanatephenol-chloroform (3). Unfortunately, for many applications, this approach is time-consuming and, more importantly, results in a significant loss of material during RNA extraction. In contrast, the use of diethyl pyrocarbonate (DEPC) water has been described as a means for rapidly determining gene expression in fresh cells, thereby eliminating an RNA extraction step (4). However, residual DEPC is undesirable because of its tendency to inhibit reverse transcription. We have therefore developed a rapid and sensitive method which can determine RNA quality in formalin-fixed, paraffin-embedded tissues. Using this procedure, tissue scraped from slides was used as a direct substrate for RT-PCR in order to amplify K-ras and protein kinase CC, (PKC-Q. Using this straightforward method, RNA template can be obtained in 1.5 h, making it feasible to reverse transcribe and amplify cDNA in a single day, a time frame appropriate for both the diagnostic and research laboratories.