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

Cecile Rose T Vibat, Michael J. Holland, John J. Kang, Luanna K. Putney, Martha E O'Donnell

Research output: Contribution to journalArticle

32 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)218-230
Number of pages13
JournalAnalytical Biochemistry
Volume298
Issue number2
DOIs
StatePublished - Dec 15 2001

Fingerprint

Polymerase chain reaction
Real-Time Polymerase Chain Reaction
Tissue
Assays
Kinetics
Cells
Messenger RNA
Genes
Cell Line
Transcription
Polymerase Chain Reaction
Protein Isoforms
Nucleotides
Throughput
Reverse Transcription
Proteins

Keywords

  • Alternative splicing
  • Brain
  • Cotransport
  • Kinetic RT-PCR
  • RNA
  • Tissue distribution
  • Trabecular meshwork

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction. / Vibat, Cecile Rose T; Holland, Michael J.; Kang, John J.; Putney, Luanna K.; O'Donnell, Martha E.

In: Analytical Biochemistry, Vol. 298, No. 2, 15.12.2001, p. 218-230.

Research output: Contribution to journalArticle

Vibat, Cecile Rose T ; Holland, Michael J. ; Kang, John J. ; Putney, Luanna K. ; O'Donnell, Martha E. / Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction. In: Analytical Biochemistry. 2001 ; Vol. 298, No. 2. pp. 218-230.
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