Implementation of exon arrays: alternative splicing during T-cell proliferation as determined by whole genome analysis.
BACKGROUND
The contribution of alternative splicing and isoform expression to cellular response is emerging as an area of considerable interest, and the newly developed exon arrays allow for systematic study of these processes. We use this pilot study to report on the feasibility of exon array implementation looking to replace the 3' in vitro transcription expression arrays in our laboratory.One of the most widely studied models of cellular response is T-cell activation from exogenous stimulation. Microarray studies have contributed to our understanding of key pathways activated during T-cell stimulation. We use this system to examine whole genome transcription and alternate exon usage events that are regulated during lymphocyte proliferation in an attempt to evaluate the exon arrays.


RESULTS
Peripheral blood mononuclear cells form healthy donors were activated using phytohemagglutinin, IL2 and ionomycin and harvested at 5 points over a 7 day period. Flow cytometry measured cell cycle events and the Affymetrix exon array platform was used to identify the gene expression and alternate exon usage changes. Gene expression changes were noted in a total of 2105 transcripts, and alternate exon usage identified in 472 transcript clusters. There was an overlap of 263 transcripts which showed both differential expression and alternate exon usage over time. Gene ontology enrichment analysis showed a broader range of biological changes in biological processes for the differentially expressed genes, which include cell cycle, cell division, cell proliferation, chromosome segregation, cell death, component organization and biogenesis and metabolic process ontologies. The alternate exon usage ontological enrichments are in metabolism and component organization and biogenesis. We focus on alternate exon usage changes in the transcripts of the spliceosome complex. The real-time PCR validation rates were 86% for transcript expression and 71% for alternate exon usage.


CONCLUSIONS
This study illustrates that the Exon array technology has the potential to provide information on both transcript expression and isoform usage, with very little increase in expense.
Whistler
Toni
T
Chronic Viral Diseases Branch, National Center for Emerging and Zoonotic Infectious Diseases (proposed), Centers for Disease Control and Prevention, Atlanta GA 30333, USA. taw6@cdc.gov
Chiang
Cheng-Feng
CF
Lonergan
William
W
Hollier
Mark
M
Unger
Elizabeth R
ER
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D016000:Cluster Analysis; D005091:Exons / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D016131:Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010835:Phytohemagglutinins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017381:Spliceosomes / Q000187:drug effects / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*
D010835:Phytohemagglutinins; D012333:RNA, Messenger