Sun Bing, Zheng Yun-Ling
Georgetown University, Washington, DC, USA.
Cancer Prevention and Control Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA.
Methods Mol Biol. 2018;1768:387-400. doi: 10.1007/978-1-4939-7778-9_22.
Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/β+, α+/β- single deletions, α-/β- double deletion, and nondeletion α+/β+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.
目前，尚无灵敏、精确且可重复的方法来定量mRNA转录本的可变剪接。液滴数字™PCR（ddPCR™）分析能够实现准确的数字计数，以定量基因表达。人端粒酶逆转录酶（hTERT）是端粒酶活性和端粒维持所需的必需成分之一。文献中已报道了人原代细胞和肿瘤细胞中hTERT mRNA的几种可变剪接形式。使用一对针对hTERT的引物和两种探针，通过一种新颖的分析方法，根据从单个ddPCR反应收集的数据，准确地定量了hTERT的四种可变剪接形式（α-/β+、α+/β-单缺失、α-/β-双缺失和非缺失α+/β+）。在本章中，我们描述了这种ddPCR方法，该方法能够直接对hTERT信使RNA的四种可变剪接形式进行定量比较，而无需内标或针对每个变体的多对引物，消除了因不同扩增子的PCR扩增效率差异导致的技术变异以及使用标准曲线进行定量的挑战。这种简单直接的方法在定量可变剪接基因转录本方面应具有普遍适用性。
