Walton Heather S, Gebhardt Florian M, Innes David J, Dodd Peter R
School of Molecular and Microbial Sciences, University of Queensland, Australia.
J Neurosci Methods. 2007 Mar 15;160(2):294-301. doi: 10.1016/j.jneumeth.2006.09.022. Epub 2006 Nov 13.
Fluorescence-based PCR techniques are becoming an increasingly popular method for measuring low-abundance alternatively spliced mRNA transcripts. The dynamic range of real-time RT-PCR affords high sensitivity for the measurement of gene expression, but this mandates the need for strict controls to ensure assay validity. Primer design, reverse transcription, and cycling conditions need to be optimized to ensure an accurate and reproducible assay. Here, we describe a procedure for creating a cost effective and reliable method for the absolute quantification of several exon-skipping variants of human excitatory amino acid transporter-2 (EAAT2). We show that the cycling conditions can be adjusted to increase the specificity of primers that span exon-exon junctions, and that differences in the reverse transcription reaction can be minimized. Standard curves are stable and produce accurate absolute copy number data. We report that exon-skipping transcripts, EAAT2Delta7 and EAAT2Delta9, account for 5.8% of EAAT2 mRNA in autopsy human neocortex.
基于荧光的PCR技术正日益成为一种用于测量低丰度可变剪接mRNA转录本的流行方法。实时逆转录PCR的动态范围为基因表达的测量提供了高灵敏度，但这就需要严格的对照来确保检测的有效性。引物设计、逆转录和循环条件都需要优化，以确保检测准确且可重复。在此，我们描述了一种用于创建一种经济高效且可靠的方法，用于绝对定量人兴奋性氨基酸转运体2（EAAT2）的几种外显子跳跃变体的程序。我们表明，可以调整循环条件以提高跨越外显子-外显子连接的引物的特异性，并且可以将逆转录反应中的差异最小化。标准曲线稳定且能产生准确的绝对拷贝数数据。我们报告称，外显子跳跃转录本EAAT2Delta7和EAAT2Delta9在尸检人类新皮层中占EAAT2 mRNA的5.8%。
