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.
Vibat
C R
CR
Department of Human Physiology, Department of Biological Chemistry, School of Medicine, University of California, One Shields Avenue, Davis, California 95616, USA.
Holland
M J
MJ
Kang
J J
JJ
Putney
L K
LK
O'Donnell
M E
ME
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005901:Glaucoma / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics*; D014018:Tissue Distribution; D014129:Trabecular Meshwork / Q000378:metabolism
D002712:Chlorides; D017931:DNA Primers; D012333:RNA, Messenger; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D011188:Potassium