Quantitation of Na+-K+-2Cl− Cotransport Splice Variants in Human Tissues Using Kinetic Polymerase Chain Reaction
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- 作者：Vibat ; Cecile Rose T. ; Holland ; Michael J. ; Kang ; John J. ; Putney ; Luanna K. ; O'Donnell ; Martha E.
- 刊名：Analytical Biochemistry
- 出版年：2001
- 出版时间：November 15, 2001
- 年：2001
- 卷：298
- 期：2
- 页码：218-230
- 全文大小：236 K
文摘
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.