Methods for enrichment of a mRNA isoform with specific alternative splicing.
Alternative splicing of pre-mRNA is an important mechanism for regulating gene expression in higher eukaryotes. Recent genomewide analyses of alternative splicing indicate that 40-60% of human genes have alternative splice isoforms, although some variants exist only in relatively low abundance. It has been shown that proteins of different functions can be produced by a diverse array of mRNA derived from a single pre-mRNA. Inevitably cloning and expression of the corresponding mRNAs (cDNA) constitute an essential step toward elucidating the function of such protein isoforms. Here, we propose methods for enriching an mRNA isoform, which is carried out before the RNA preparation is subjected to reverse transcription polymerase chain reaction and cloning. While the negative selection method destroys unwanted mRNA variants, the positive selection enriches the desired variant. Focusing on the mRNA of rat ADAR2 (adenosine deaminase 2 that acts on RNA) as an example, we have achieved 16- and 19-fold enrichment of the given mRNA variant via the proposed negative and positive selection, respectively. Single use or combined uses of our method facilitates the isolation and cloning of a minor mRNA variant. Moreover, these methods can also be used to determine whether two alternative splicing events taking place in a pre-mRNA are linked.
Wang
Nai-Yu
NY
Graduate Institute of Medical Technology, National Yang-Ming University, Taipei 112, Taiwan, Republic of China.
Lai
Hsiu-Chun
HC
Au
Lo-Chun
LC
D000243:Adenosine Deaminase; D017398:Alternative Splicing; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D005091:Exons / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D020033:Protein Isoforms; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase