Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction.
Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction.
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使用动态聚合酶链反应定量人体组织中的 Na -K -2Cl- 共转运剪接变体。
DOI：
10.1006/abio.2001.5398
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
O'Donnell,ME
中科院分区：
文献类型：
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作者：
Vibat,CR;Holland,MJ;Kang,JJ;Putney,LK;O'Donnell,ME
描述了一种基于动态逆转录聚合酶链式反应(RT-PCR)的方法，可以区分和定量差异剪接的mRNAs。该方法应普遍适用于差异剪接转录本的高通量定量。对由人Na+-K+-2ClhNKCC1共转运蛋白基因编码的剪接转录本的实用性进行了评估。有证据表明，人类Na+-K+-2CL−共转运蛋白的NKCC1亚型与最近描述的小鼠Na+-K+-2CL−共转运蛋白基因BSC2的剪接有差异。这两个人类剪接变异体的核苷酸序列预测了Na+-K+-2Cl−共转运蛋白，只是长度不同。构建了稳定表达这些人剪接变异体的转染体，命名为NKCC1a或NKCC1b。这两种剪接变异体在体内都能产生功能性的Na+-K+-2Cl−共转运蛋白。采用KRT-PCR方法检测NKCC1在10种不同组织类型和3种细胞系中的丰度和差异剪接模式。结果表明，在所研究的人体组织和细胞系中，NKCC1mRNA的总量变化了30倍以上。在这些相同的组织和细胞系中，NKCC1a/NKCC1b的比率变化了近70倍，这表明NKCC1转录本的差异剪接可能在人类组织中发挥调节作用。
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.