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

Anal Biochem. 2001 Nov 15;298(2):218-30. doi: 10.1006/abio.2001.5398.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing / genetics*
  • Amino Acid Sequence
  • Base Sequence
  • Cells, Cultured
  • Chlorides / metabolism
  • DNA Primers / chemistry
  • Glaucoma / metabolism
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Potassium / metabolism
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Sequence Homology, Nucleic Acid
  • Sodium / metabolism
  • Sodium-Potassium-Chloride Symporters / genetics*
  • Tissue Distribution
  • Trabecular Meshwork / metabolism

Substances

  • Chlorides
  • DNA Primers
  • RNA, Messenger
  • Sodium-Potassium-Chloride Symporters
  • Sodium
  • Potassium