Identification of low-abundance alternatively spliced mRNA variants by exon exclusive reverse transcriptase...

Alternative splicing of messenger RNA (mRNA) precursors generates multiple transcripts from a single primary transcript. Identification and verification of splice variants and cloning of the corresponding isoforms is crucial for analyzing gene expression and understanding the related functions. For a specific gene, the abundance of the transcripts produced can vary significantly and is subject to various regulations. It can be difficult to detect low-level splicing variants when others are present in high abundance. Here we describe a method for the amplification of low-abundance mRNA splicing variants for such situations. This method introduces a hydrolysis step prior to the conventional reverse transcriptase polymerase chain reaction (RT-PCR). After the transcripts are reverse-transcripted into complementary DNA (cDNA), the cDNA of high-abundance transcript is suppressed from amplification by cleavage at the chosen exon to enhance the amplification of the low-abundance transcripts that do not have the targeted exon and are normally undetectable. We provide two examples to illustrate the detection of low-abundance splicing variants from two genes.