Quantitation of Na⁺-K⁺-2Cl^- cotransport splice variants in human tissues using kinetic polymerase chain reaction

A kinetic reverse transcription-polymerase chain reaction (RT-PCR)-based assay is described that can discriminate and quantitate differentially spliced mRNAs. This assay should be generally applicable for high-throughput quantitation of differentially spliced transcripts. The utility of this method was assessed for spliced transcripts encoded by the human Na⁺-K⁺-2Cl^- cotransporter gene hNKCC1. Evidence is presented that the NKCC1 isoform of the human Na⁺-K⁺-2Cl^- cotransporter is differentially spliced analogous to that recently described for the mouse Na⁺-K⁺-2Cl^- cotransporter gene BSC2. The nucleotide sequences of the two human splice variants predict Na⁺-K⁺-2Cl^- cotransporter proteins differing only in length. Stable transfectants expressing these human splice variants, designated NKCC1a or NKCC1b, were constructed. Both splice variants produce functional Na⁺-K⁺-2Cl^- cotransporters in vivo. The abundance of NKCC1 mRNA and patterns of differential splicing in 10 different tissue types and three cell lines were quantitated using the kRT-PCR assay. The results showed that the total amount of NKCC1 mRNA varied by more than 30-fold in the human tissues and cell lines examined. The ratio of NKCC1a/NKCC1b varied nearly 70-fold among these same tissues and cell lines suggesting that differential splicing of the NKCC1 transcript may play a regulatory role in human tissues.