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 language | English (US) |
|---|---|
| Pages (from-to) | 218-230 |
| Number of pages | 13 |
| Journal | Analytical Biochemistry |
| Volume | 298 |
| Issue number | 2 |
| DOIs | |
| State | Published - Dec 15 2001 |
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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 journal › Article
}
TY - JOUR
T1 - Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction
AU - Vibat, Cecile Rose T
AU - Holland, Michael J.
AU - Kang, John J.
AU - Putney, Luanna K.
AU - O'Donnell, Martha E
PY - 2001/12/15
Y1 - 2001/12/15
N2 - 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.
AB - 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.
KW - Alternative splicing
KW - Brain
KW - Cotransport
KW - Kinetic RT-PCR
KW - RNA
KW - Tissue distribution
KW - Trabecular meshwork
UR - http://www.scopus.com/inward/record.url?scp=0035892605&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035892605&partnerID=8YFLogxK
U2 - 10.1006/abio.2001.5398
DO - 10.1006/abio.2001.5398
M3 - Article
C2 - 11700976
AN - SCOPUS:0035892605
VL - 298
SP - 218
EP - 230
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -