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Analyses of Affymetrix exon array to investigate
alternative splicing in human brain
Amandine Bemmo1,2, David Benovoy2, Jacek
Majewski21Universite de Montreal, 2McGill
university and Genome Quebec innovation centre
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 Alzheimers 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.
Exemples of isoform differences
A

• OBJECTIVES
• 1. Compare and contrast alternative splicing
  between human brain and human reference tissues
• Identify new transcript isoforms
• Develop an analysis protocol for the Human Exon
  Array

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.
EXPERIMENTAL DESIGN
Categorization of isoform differences
Analysis
List of different tissues that comprise the Human
Brain reference and the Human reference
Extraction of RNAs samples (Brain and reference)
for chip hybridization. Microarray datas are
processed and expression levels are determinated.
10 replicates for each sample.
ANALYSIS PROTOCOL

• Flow chart for the identification of Alternative
  splicing events
• 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.
• Presence or absence of exons is determined by
  using the detection above background metric
  (DABG).
• Filter The estimated p-value cutoff of
  0.01859597 (FDR lt 0.05) yields to a set of
  significant probesets to decrease the rate of
  false positives and to obtain meaningful splicing
  information.
• The data are visualized with UCSC genome browser
  in the context of gene structure and known
  isoform (EST-based) information.
• Isoform event types are categorized alternative
  splicing, alternative promoter usage, alternative
  termination.
• Validation of candidate exons is done by using
  RT-PCR

Categorization of different isoform events
between brain and the reference tissues.
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.

• FUTHER WORKS
• Catalogue brain specific isoforms
• Find the binding sequences of alternative
  splicing factors.
• Develop an analysis protocol for the Human Exon
  Array.

RESULTS Transcript and exon differences

• REFERENCES
• Affymeytrix Identifying and Validating
  Alternative Splicing Events An introduction to
  managing data provided by GeneChip Exon Arrays.
  Affymetrix Technical Note
• Paul J Gardina et al. Alternative splicing and
  differential gene expression in colon cancer
  detected by a whole genome exon array. BMC
  Genomics 2006, 7325
• Tyson A Clark et Al. Discovery of
  tissue-specific exons using comprehensive human
  exon microarrays. Genome Biologie 2007, 8R64

Number of significant transcript differences Number of significant exons differences
8,794 43,375
From a potential 17,919 core transcripts From
a potential 188,931 core exons