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Nucleic Acids Research Advance Access originally published online on August 11, 2009
Nucleic Acids Research 2009 37(19):e126; doi:10.1093/nar/gkp626
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Nucleic Acids Research, 2009, Vol. 37, No. 19 e126
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Analysis of splicing patterns by pyrosequencing

Agnès Méreau1,2,3, Vincent Anquetil1,2,3, Marie Cibois1,2,3, Maud Noiret1,2,3, Aline Primot1,3,4, Audrey Vallée1,3,4 and Luc Paillard1,2,3,*

1Institut de Génétique et Développement de Rennes, Université de Rennes 1, IFR 140, 2CNRS, UMR6061, équipe Expression Génétique et Développement, 3Université Européenne de Bretagne and 4CNRS, UMR6061, équipe Régulation Transcriptionnelle et Oncogenèse, F-35000 Rennes, France

*To whom correspondence should be addressed. Tel: +33 223 23 44 73; Fax: +33 223 23 44 78; Email: luc.paillard{at}univ-rennes1.fr

Received June 11, 2009. Revised July 12, 2009. Accepted July 13, 2009.

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.


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