SELECTIVE ENZYMATIC DEPLETION DURING RNA-PCR - IMPROVED DETECTION OF RARE RNA FORMS

A procedure that facilitates the detection of rare RNAs by RNA-polymer ase chain reaction (RNA-PCR) when a large excess of competing template is present in the PCR is described. We have used this assay to detect spliced mRNAs of low abundance, which result from in vitro splicing o f an intron derived from the rat fibronectin gene. RNA was isolated fr om splicing reactions, reverse transcribed, then PCR was performed, us ing primers based on exon sequences. After a limited number of cycles, amplified DNAS were incubated with a restriction enzyme which cleaved only within the intron, leaving the spliced two-exon product intact. This step selectively depleted the unspliced pre-mRNA, and subsequent rounds of PCR served to enrich for spliced product. The spliced produc t was not detected by performing PCR without restriction digestion to deplete pre-mRNA-derived species. The broader application of the enzym atic depletion strategy to detection of rare alternative mRNA isoforms is also demonstrated.