Analysis of splicing patterns by pyrosequencing.
Several different mRNAs can be produced from a given pre-mRNA by regulated alternative splicing, or as the result of deregulations that may lead to pathological states. Analysing splicing patterns is therefore of importance to describe and understand developmental programs, cellular responses to internal or external cues, or human diseases. We describe here a method, Pyrosequencing Analysis of Splicing Patterns (PASP), that combines RT-PCR and pyrosequencing of PCR products. We demonstrated that: (i) Ratios of two pure RNAs mixed in various proportions were accurately measured by PASP; (ii) PASP can be adapted to virtually any splicing event, including mutually exclusive exons, complex patterns of exon skipping or inclusion, and alternative 3' terminal exons; (iii) In extracts from different organs, the proportions of RNA isoforms measured by PASP reflected those measured by other methods. The PASP method is therefore reliable for analysing splicing patterns. All steps are done in 96-wells microplates, without gel electrophoresis, opening the way to high-throughput comparisons of RNA from several sources.
Méreau
Agnès
A
Institut de Génétique et Développement de Rennes, Université de Rennes 1, IFR 140, CNRS, UMR6061, Equipe Expression Génétique et Développement, Université Européenne de Bretagne, F-35000 Rennes, France.
Anquetil
Vincent
V
Cibois
Marie
M
Noiret
Maud
M
Primot
Aline
A
Vallée
Audrey
A
Paillard
Luc
L
D017398:Alternative Splicing; D000818:Animals; D005091:Exons; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D014981:Xenopus