QUANTIFICATION OF ALTERNATIVELY SPLICED RUSH MESSENGER-RNA ISOFORMS BY QRT-PCR AND IP-RP-HPLC ANALYSIS - A NEW APPROACH TO MEASURING REGULATED SPLICING EFFICIENCY

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and propor tional quantification of alternatively spliced RUSH mRNAs. RUSH isofor ms result from alternative splicing of a 57-bp exon and encode SNF/SWI -related proteins that bind to the uteroglobin promoter. QRT-PCR was p erformed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPL C identified the specific homoduplex products, as well as the heterodu plexes formed as a consequence of partial sequence complementarity bet ween the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes i n the relative levels of RUSH message isoforms. The preferential expre ssion of the RUSH-1 alpha isoform by all the tissues except estrous ut erine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold diff erence in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endome trium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and ver satile tools for the detection and quantitation of mRNA splice variant s. (C) 1997 Elsevier Science B.V.