OPTIMIZATION OF EXTRACTION AND PCR AMPLIFICATION OF RNA EXTRACTS FROMPARAFFIN-EMBEDDED TISSUE IN DIFFERENT FIXATIVES

A method was developed for fast and efficient isolation of RNA from pa raffin-embedded tissue sections for subsequent PCR analysis. This meth od is based on the binding of RNA to acid-treated glass beads in the p resence of a high molarity of guanidinium salt. It can be completed wi thin an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was t ested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the e xtracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polyme rase chain reaction amplification (RT-PCR), using virus-infected and m ock-infected paraffin-embedded cell pellets as a model. PCR amplificat ion of cellular and viral RNA was successful for RNA isolated by use o f all extraction techniques, although the glass bead method was prefer red for its simplicity and rapidity. Specimens fixed with formalin wer e found to be suitable for PCR, but the best results were obtained wit h acetone-fixed paraffin-embedded material. Dewaxing of tissue section s had no effect on the yield and quality of RNA extractions, and furth er purification of the extracts using gel filtration did not improve t he results. After the protocols were optimized, rotavirus-infected cel l pellets were used to demonstrate that extraction and amplification o f dsRNA was possible. The information obtained from the studies with t he model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin- embedded archival tissue can be used for RT-PCR analysis, adding an im portant technique to diagnostic pathology and retrospective studies.