A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples
A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples
复制标题
DOI：
10.1093/nar/gkm989
复制
发表时间：
2007-12-01
影响因子：
14.9
通讯作者：
Mitra, Robi David
中科院分区：
文献类型：
--
作者：
Leparc, German Gaston;Mitra, Robi David
基因组学的一个重要目标是探索转录组中可变剪接的程度，并产生一个全面的剪接形式目录。新的计算和实验方法导致预测的选择性剪接转录本的数量增加;然而，这些预测的验证并没有跟上。在这项工作中，我们系统地探讨了不同的方法来验证盒外显子预测的计算方法或平铺微阵列。我们的目标是找到一种具有成本效益，敏感和具体的程序。我们研究了逆转录反应的三种启动方式：poly-dT启动、随机启动和混合外显子特异性启动。我们还研究了两种策略的PCR amplificationflanking PCR，它使用的引物，杂交的组成型外显子侧翼预测的外显子，和一个半巢式PCR的引物，目标预测的外显子。我们发现，使用基因特异性引物池的RT与半巢式PCR的组合导致灵敏度比最常用的方法显著增加（检测到97%的测试集对14%）。我们的方法也是高度特异性的，使用真阴性的测试集没有检测到假阳性。最后，我们证明了这种方法能够从整个生物体RNA中以高灵敏度检测替代外显子，允许在单个实验中对所有组织进行采样。这里开发的协议是一个准确和具有成本效益的方式来验证选择性剪接的预测。
One important goal of genomics is to explore the extent of alternative splicing in the transcriptome and generate a comprehensive catalog of splice forms. New computational and experimental approaches have led to an increase in the number of predicted alternatively spliced transcripts; however, validation of these predictions has not kept pace. In this work, we systematically explore different methods for the validation of cassette exons predicted by computational methods or tiling microarrays. Our goal was to find a procedure that is cost effective, sensitive and specific. We examined three ways of priming the reverse transcription (RT) reactionpoly-dT priming, random priming and pooled exon-specific priming. We also examined two strategies for PCR amplificationflanking PCR, which uses primers that hybridize to the constitutive exons flanking the predicted exon, and a semi-nested PCR with a primer that targets the predicted exon. We found that the combination of RT using a pool of gene-specific primers followed by semi-nested PCR resulted in a significant increase in sensitivity over the most commonly used methodology (97% of the test set was detected versus 14%). Our method was also highly specificno false positives were detected using a test set of true negatives. Finally, we demonstrate that this method is able to detect alternative exons with a high sensitivity from whole-organism RNA, allowing all tissues to be sampled in a single experiment. The protocol developed here is an accurate and cost-effective way to validate predictions of alternative splicing.