1. Amandine Bemmo 1,2, David Benovoy 2, Jacek Majewski 2 1 Universite de Montreal, 2 McGill university and Genome Quebec innovation centre Analyses of Affymetrix exon array to investigate alternative splicing in human brain ABSTRACT Alternative splicing (AS) is a process that generates multiple isoforms from the same mRNA precursor and increases proteomic diversity. AS plays a critical role in regulating brain processes. Variation in AS among individuals may be responsible for phenotypic diversity and differential susceptibility to genetic disorders such as Alzheimer´s disease, spinal muscular atrophy, frontotemporal dementia and certain malignancies. The Affymetrix Human Exon Array, that target individual exons, was used to compare AS in human brain and human reference tissues. We identified many known (EST-based and RNA-based) and novel cases of AS that that vary between human brain and human reference tissue. OBJECTIVES 1. Compare and contrast alternative splicing between human brain and human reference tissues 2.Identify new transcript isoforms 3.Develop an analysis protocol for the Human Exon Array EXON ARRAY DESIGN The Affymetrix Human Exon 1.0 ST array was used for this study. Show to the left is the design of probes on the array. A genomic locus comprises exons (yellow) and introns (grey). Probe set (red) corresponding to each exon are represented by 25-mer probes. The array features 5.5x10 6 probes, corresponding to 1x10 6. FUTHER WORKS 1.Catalogue brain specific isoforms 2.Find the binding sequences of alternative splicing factors. 3.Develop an analysis protocol for the Human Exon Array. CONCLUSION Affymetrix exon array can detect a variety of annotated and novel splicing events in human brain, demonstrating it is potential as a discovery tool and increasing the catalogue of human transcriptome. Early analyses show that alternative splicing is very frequent in brain and plays a key role in brain complexity. We are doing more analyses to explain and understand the role of alternative splicing in brain. EXPERIMENTAL DESIGN Analysis Exemples of isoform differences A. Alternative initiation in OSBPL1A (oxysterol-binding protein-like 1A): BC098334 (green box) is an isoform of gene OSBPL1A present in human brain. The pink boxes represent alternative promoter usage between brain and reference transcript. The fold chanqes (brain/reference) in blue of exons expression of the affymetrix exon array support this event. B. Alternative cassette exon in RAB6IP2 (RAB6-interacting protein 2): The isoform of RAB6IP2 present in brain is AB029004 (surrounded in geen); it is an example of alternative splicing (surrounded in pink). C. Alternative 5’ splice site and cassette exon in STAU (staufen): AB209561 (green box) is the isoform of gene STAU present in brain. Alternative 5’ splice site and exon cassette are surrounded in pink. In the second event (the most rigth), the exon is skipped in reference and included in brain; the foldchange of this exon validate this event. B C ANALYSIS PROTOCOL Flow chart for the identification of Alternative splicing events 1.Exon signal and gene signal are performed with PLIER algorithm which summarize the probe signals for exon signal and the probeset signals for gene signal. 2.Presence or absence of exons is determined by using the detection above background metric (DABG). 3.Filter: The estimated p-value cutoff of 0.01859597 (FDR < 0.05) yields to a set of significant probesets to decrease the rate of false positives and to obtain meaningful splicing information. 4.The data are visualized with UCSC genome browser in the context of gene structure and known isoform (EST-based) information. 5.Isoform event types are categorized: alternative splicing, alternative promoter usage, alternative termination. 6.Validation of candidate exons is done by using RT-PCR A Categorization of isoform differences REFERENCES 1.Affymeytrix: Identifying and Validating Alternative Splicing Events: An introduction to managing data provided by GeneChip® Exon Arrays. Affymetrix Technical Note 2.Paul J Gardina et al.: Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics 2006, 7:325 3.Tyson A Clark et Al.: Discovery of tissue-specific exons using comprehensive human exon microarrays. Genome Biologie 2007, 8:R64 RESULTS Transcript and exon differences Number of significant transcript differences * Number of significant exons differences** 8,79443,375 *From a potential 17,919 core transcripts **From a potential 188,931 core exons Categorization of different isoform events between brain and the reference tissues. List of different tissues that comprise the Human Brain reference and the Human reference Extraction of RNA’s samples (Brain and reference) for chip hybridization. Microarray datas are processed and expression levels are determinated. 10 replicates for each sample.