Sensitive detection of mRNA decay products by use of reverse-ligation-mediated PCR (RL-PCR).
Ligation-mediated PCR allows the detection and mapping of cleavage products of specific nucleic acid molecules out of complex nucleic acid mixtures. It can be applied to the detection of either degradation products of exogenously added nucleases in footprinting applications or natural decay products or reaction intermediates. We have developed various ligation-mediated PCR approaches to analyze mRNAs, all relying on RNA ligation, followed by reverse-transcription and PCR amplification. We have termed these approaches reverse-ligation-mediated PCR (RL-PCR). The ligation event involves either an RNA linker added to the 5'-end of cleaved RNA or RNA circularization, allowing, respectively, the mapping and quantification of the cleavage points or the simultaneous analysis of the presence or absence of the 5'-cap structure and the length of the poly(A) tail. These methods enabled us to develop a very efficient 5'-RACE procedure to map mRNA 5'-ends, to footprint in permeabilized cells the interaction of regulatory proteins with RNA, to detect the products of cellular ribozyme action and to analyze cellular decay pathways that involve deadenylation and/or decapping. I review herein the methodologic aspects and protocols of the various RL-PCR procedures we have developed.
Grange
Thierry
T
Institut Jacques Monod du CNRS, Université Paris 7, Paris, France.
D000818:Animals; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D011506:Proteins; D012333:RNA, Messenger