Selective enzymatic depletion during RNA-PCR: improved detection of rare RNA forms.
A procedure that facilitates the detection of rare RNAs by RNA-polymerase chain reaction (RNA-PCR) when a large excess of competing template is present in the PCR is described. We have used this assay to detect spliced mRNAs of low abundance, which result from in vitro splicing of an intron derived from the rat fibronectin gene. RNA was isolated from splicing reactions, reverse transcribed, then PCR was performed, using primers based on exon sequences. After a limited number of cycles, amplified DNAs were incubated with a restriction enzyme which cleaved only within the intron, leaving the spliced two-exon product intact. This step selectively depleted the unspliced pre-mRNA, and subsequent rounds of PCR served to enrich for spliced product. The spliced product was not detected by performing PCR without restriction digestion to deplete pre-mRNA-derived species. The broader application of the enzymatic depletion strategy to detection of rare alternative mRNA isoforms is also demonstrated.
Norton
P A
PA
Department of Medicine, Roger Williams Medical Center and Brown University, Providence, RI 02908.
McInnis
R
R
D000818:Animals; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000032:analysis*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D013698:Templates, Genetic; D014982:Xenopus laevis
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; C064060:endodeoxyribonuclease EcoNI; C045874:endodeoxyribonuclease StyI; D015252:Deoxyribonucleases, Type II Site-Specific