Medium throughput analysis of alternative splicing by fluorescently labeled RT-PCR

Abstract

Reverse transcription-PCR (RT-PCR) is a core technique for detecting and quantifying alternative pre-mRNA splicing. RT-PCR is multistep process involving RNA isolation, reverse transcription, and PCR that is often performed using radiolabeled primers. As a result RT-PCR analysis of alternative splicing is a laborious technique that quickly becomes prohibitively expensive when applied to large numbers of samples. Here, we describe an RT-PCR approach for detecting alternative splicing in multi-well plates that can be applied to effortlessly quantify exon inclusion levels in large number of samples. The procedures outlined here can also be automated on standard liquid handling equipment to produce medium throughput assay capable of handling thousands of samples per day. © 2014 Springer Science+Business Media, LLC.