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L6_OneDyeGeneExp - ECEN 689 Statistical Computation in GSP...

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ECEN 689 Statistical Computation in GSP http://www.ece.tamu.edu/~ulisses/ECEN689/ Lecture 6: One-Dye Gene-Expression Microarrays Ulisses Braga Neto Genomic Signal Processing Laboratory Department of Electrical and Computer Engineering Texas A&M University
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Gene-Expression Microarray Three main gene-expression microarray technologies: – cDNA, ~400-base cloned probes (old) – One-color oligonucleotide, 25-base sequenced probes (Affymetrix) – Two-color oligonucleotide, 25- to 60-base sequenced probes (Agilent, Amersham)
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Affymettrix GeneChip
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Photolithographic Process Lamp Mask Wafer
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Probe Synthesis HO HO O O O Light (deprotection) Mask O O O O O Substrate Light (deprotection) T T O O O T T O O O T – T T C C O C – C A T A T A G C T G T T C C G Continue
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Feature Size Features/Chip 100 μm 50 μm 24 μm 20 μm 18 μm 11 μm 16,384 65,538 284,444 409,600 506,944 >1,350,000 Genes/Chip 409 1,638 7,111 12,800 22,500 54,000 High-Density Chips For fixed array size, 1.28 x 1.28 cm
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Probe Array Overview * * * * * *
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Human Whole Genome Arrays HG-U133 2.0 Plus - 11um features - 54000 probe sets (probes have a 3’ end bias) - 6500 more genes than U133 set - entire content on one chip Human Gene 1.0 ST Array - 5 um features - 28869 probe sets (probes spread through gene)
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Gene Sequence Multiple 25-mer oligo probes
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Perfect Match (PM) Mismatch (MM) Probe Pair Probe Set TACGACCGCTAATCGTAAATGTCAG TACGACCGCTAA A CGTAAATGTCAG
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Assay Overview Process Control
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Poly-A control kit Manufactured by Ambion for Affymetrix Poly-A Control Stock: Lys, Phe, Thr, and Dap at staggered concentrations (i.e. complexity ratios). • Lys at 1:100,000 • Phe at 1:50,000 • Thr at 1:25,000 • Dap at 1:7,500 Poly-A Control Dilution Buffer Product Insert contains dilution instructions Material sufficient for about 100 reactions
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Target Amplification One or two-cycle target amplification
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One-cycle cDNA Synthesis
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Two-cycle cDNA Synthesis
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Sample Cleanup and IVT
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In Vitro Transcription Kit Proprietary biotinylated nucleotide analog Ambion Megascript based Start with as low as 1ug total RNA 30 reactions Over night incubation or 4hr with enzyme spike-in
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Hybridization/Scanning Scanned Image Scan Scanner Wash & Stain Fluidics Station Hybridize Hybridization Oven
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Data Files .DAT : Raw scanned images (big) Each probe cell corresponds to a square of 64x64 pixels .CEL : Summarizes the info in the .DAT image by one single value (average intensity) for each probe cell. Much smaller file and point of start of most analyses. .CDF : Specific to each chip, contains addressing info for each probe set. .CHP : contains summary information of the probe sets, including intensity values. .EXP : contains information about experimental conditions and protocols
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Quality-Control Measures Visual Array Inspection Grid Alignment (oligo b2) poly-A control spikes dap, lys, phe, thr, trp Hybridization control spikes bioB, bioC, bioD, Cre House-keeping genes % present Scaling Factor Q Noise Factor • Background • Histograms/Scatterplots
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