Gene Expression BMI 731 Winter 2005. Catalin Barbacioru Department of Biomedical Informatics Ohio State University. Thesis: the analysis of gene expression data is going to be big in 21st century statistics. Many different technologies, including Spotted DNA arrays (Brown/Botstein)
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Department of Biomedical Informatics
Ohio State University
Many different technologies, including
Spotted DNA arrays (Brown/Botstein)
Short oligonucleotide arrays (Affymetrix)
Serial analysis of gene expression (SAGE)
Long oligo arrays (Agilent)
Fibre optic arrays (Illumina)
Number of papers
1995 1996 1997 1998 1999 2000 2001
Total microarray articles
indexed in Medline
Idea: measure the amount of mRNA to see which genes are being expressedin (used by) the cell.
Measuring protein might be better, but is currently harder.
Platform for GeneChip® Probe Arrays
Light determining the complete expression profile of a cell.
HO HO OOO
REPEATSynthesis of Ordered Oligonucleotide Arrays
* determining the complete expression profile of a cell.
*GeneChip® Probe Arrays
Hybridized Probe Cell
labeled RNA target
Millions of copies of a specific
Image of Hybridized Probe Array
Each pixel is quantitated and integrated for each oligo feature (range 0-25,000)
Perfect Match (PM)
Mis Match (MM) Control
log(PM / MM) = difference score
All significant difference scores are averaged to create “average difference” = expression level of the gene.
Analysis of expression level from probe sets determining the complete expression profile of a cell.
• each oligo sequence (20-25 mer) is synthesized
as a 20 µ square (feature)
• each feature contains > 1 million copies of the oligo
• scanner resolution is about 2 µ (pixel)
• each gene is quantitated by 16-20 oligos and
compared to equal # of mismatched controls
• 22,000 genes are evaluated with 20 matching oligos
and 10 mismatched oligos = 480,000 features/chip
• 480,000 features are photolithographically synthesized onto a 2 x 2 cm glass substrate
Top 2.5%of ratios red, bottom 2.5% of ratios green
- highly reliable: synthesized in situ
- highly reproducible from run to run
- no clone maintenance or ‘drift’
- sealed fluidics and controlled temperature
- standardized chips increase database power
- excellent scanner
- complex, but very reliable labelling
- excellent cost/benefit ratio
- amenable to mutation and SNP detection
Biological question determining the complete expression profile of a cell.
Differentially expressed genes
Sample class prediction etc.
16-bit TIFF files
(Rfg, Rbg), (Gfg, Gbg)