Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

Abstract

BackgroundMicroarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events.ResultsThe array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer.ConclusionsThis methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

Putative Prostate Cancer Risk SNP in an Androgen Receptor‐Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

“…Dysregulation of melanophilin was found in several types of tumors, e.g. lung cancer, meningiomas and breast cancer (Fevre-Montange, et al, 2009; Molina-Pinelo, et al, 2014; Pio, et al, 2010; Thakkar, et al, 2010). A very recent study found association of expression of MLPH (and some other genes) with nearby SNPs in prostate tissue (Penney, et al, 2015).…”

A network-based analysis of colon cancer splicing changes reveals a tumorigenesis-favoring regulatory pathway emanating from ELK1

“…Our analysis could therefore determine additional regulatory targets (see Supplemental Discussion). To this end, we compiled a list of 54 experimentally validated alternative splicing events shown to undergo a splicing pattern change in colon tumor samples compared with corresponding normal tissue samples (Jordan et al 1999;Hardt et al 2003;Ghigna et al 2005;Gardina et al 2006;Klinck et al 2008;Thorsen et al 2008;Venables et al 2009;Langer et al 2010;Misquitta-Ali et al 2010;Pio et al 2010;Choi et al 2011;Ishimoto et al 2011;Liu et al 2012;Miura et al 2012;Seo et al 2012). We then identified the potential regulators of these cancerous splicing changes by the number and strength of determined Pearson correlations between the splicing changes of the 54 alternative events across 48 human tissues and cell lines and the gene expression of alternative splicing factors in the same tissues (Methods).…”

“…The Pearson correlations between the splicing profiles of 54 cancer-associated alternative splicing events (Jordan et al 1999;Hardt et al 2003;Ghigna et al 2005;Gardina et al 2006;Klinck et al 2008;Thorsen et al 2008;Venables et al 2009;Langer et al 2010;Misquitta-Ali et al 2010;Pio et al 2010;Choi et al 2011;Ishimoto et al 2011;Liu et al 2012;Miura et al 2012;Seo et al 2012) across all tissues and the gene expression profiles of alternative splicing factors (gene list derived from Manley 2009 andHegele et al 2012) in the same tissues were computed (alternative splicing and gene expression data were derived from Castle et al 2008). Alternative splicing factor genes not displaying a significant expression level change in colon tumors compared with a mixed tissue pool were discarded as were splicing events with significant isoform changes compared with a mixed tissue pool in only four tissues or less, and statistically insignificant correlations (P > 0.05).…”

Identification of Alternative Splicing Events Regulated by the Oncogenic Factor SRSF1 in Lung Cancer

“…The former were developed by Oryzon Genomics in collaboration with our group, and have been previously described (27). These arrays contain exon probes and thermodynamically balanced junction probes.…”

“…These high-density arrays contain an average of 8 probes per probeset. RNA labeling and hybridization on the Agilent microarrays were performed by Oryzon Genomics, as previously described (27). Labeling and hybridization on the Affymetrix GeneSplice microarrays were performed by the Proteomics, Genomics and Bioinformatics Core Facility of the Center for Applied Medical Research (CIMA) following manufacturer's instructions.…”