Identification of low-abundance alternatively spliced mRNA variants by exon exclusive reverse transcriptase polymerase chain reaction.
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.
Wang
Feng
F
State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.
Zhao
Yong
Y
Hao
Yuhua
Y
Tan
Zheng
Z
D017398:Alternative Splicing / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019014:fas Receptor / Q000235:genetics
D018076:DNA, Complementary; C491901:FAS protein, human; D012333:RNA, Messenger; D019014:fas Receptor