Quantification of alternatively spliced RUSH mRNA 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 proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed 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-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.