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This article is part of the supplement: Proceedings of the 5th International Conference of the Brazilian Association for Bioinformatics and Computational Biology (X-meeting 2009)

Open Access Proceedings

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

Elisa N Ferreira12, Maria CR Rangel1, Pedro F Galante3, Jorge E de Souza3, Gustavo C Molina1, Sandro J de Souza3 and Dirce M Carraro1*

Author Affiliations

1 Laboratory of Genomics and Molecular Biology, Hospital A.C. Camargo, Fundação Antonio Prudente, São Paulo, 01509-900, Brazil

2 Department of Genetics and Evolutionary Biology, Institute of Biosciences University of São Paulo, São Paulo, 05508-090, Brazil

3 Laboratory of Computational Biology, Ludwig Institute for Cancer Research, São Paulo, 01323-903, Brazil

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BMC Genomics 2010, 11(Suppl 5):S4  doi:10.1186/1471-2164-11-S5-S4

Published: 22 December 2010

Abstract

Background

Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression.

Results

The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system.

Conclusions

In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.