1Microarrays for Gene Expression AnalysisQuestions: What genes are expressed in this tissue under theseconditions?What genes are expressed in my treated cells versus the control?What genes are expressed during the phases of the cell cycle?What genes are expressed in diseased tissue versus normal tissue?Microarrays – other usesQuestions: What point mutations exist and what bases are located at the substitution positions?What bases are substituted where there are multiple mutations very close together?Which allele of this gene do we have?Is this the mutant or wildtype?GoalsFinding Co-Regulated GenesUnderstanding Gene Regulatory NetworksExpressed Genes = mRNADNA messenger RNA protein Expressed Genes = Currently TranscribedmRNAExtract RNAmRNAmRNAmRNAmRNAIsolate mRNA’sAffymetrix Oriented• Fluorescently tagged cRNA • One chip per sample• One for control• One for each experiment• Other methods include two dyes/one chip•Red dye• Green dye• Control and experiment on same chip2Creating TargetsmRNAReverse TranscriptasecDNAPCR Amplificationof DNAIn Vitro transcription to create cRNARNA-DNA Hybridizationprobe setsDNA(25 base oligonucleotides of known sequence)TargetsRNANon-Hybridized Targets are Washed Away“probe sets” (oligo’s)Targets(fluorescently tagged)Non-bound ones are washed awayPicture of Gene ChipHandling Chip3Argon laser488nm570nmScanner based on epifluorescence confocal microscopyCustom Chips vs Affy Chips• Affy chips contains thousands of gene probes• Genes selected from sources such as GenBank• Custom chips can be designed for individual investigators• Few genes, but more copies of each• Done on microscope slideExample Affy Chips• Rat Toxicology Chip - >850 genes• CYP450’s, Heat Shock proteins• Drug transporters• Stress-activated kinases• Rat Neurobiology chip - > 1,200 genes•Synuclein 1, prion protein, Huntington’s disease• Syntaxin, Neurexin, neurotransmittersExample Affy Chips• Arabidopsis Genome Chip• Murine Genome Chip - >36,000 genes• E. coli Genome Chip - >4,200 ORF’s• Drosophila Genome Chip - >13,500 sequences• Yeast Genome Chip - >6,400 ORF’s• Human Genome Chip - >>60,000 human genes DefinitionsProbe – a single-stranded DNA oligonucleotidecomplementary to a specific sequence. Each probe cell consists of millions of probe molecules.Probe Array – a collection of probes sets.Probe Set – a set of probes designed to detect one transcript. 16-20 probe pairs. A 20 probe pair set is made up of 20 PM and 20 MM for a total of 40 probe cells.Probe Pair – Two probe cells, a PM and its corresponding MM.Perfect Match(PM) – probes that are designed to be complementary to the reference sequence.MisMatch(PM) – probes that are designed to be complementary to the reference sequence except for 1 base.Target – sequence from your sample.GeneChip HierarchyProbe Array = ChipProbe Set – 16-20 probe pairs(to detect particular gene)Probe Pair Probe Cell (MisMatch) 20Probe Cell (Perfect Match) 20Probes <= 25 bases (millions of copies)Pixels 24 sq. um4Probe Set 1probe cellprobe pairProbe Array (chip)Probe Set 2Probe Set 3PMMMProbe PairEach pair represents a different subsequence of the genePMMMMMProbe CellProbe SetProbe – a single-stranded DNA oligonucleotide complementary to a specific sequence. Each probe cell consists of millions of the same probe molecules.The intensity of each cell is an average of each of its scanned pixels.Probe Cell20 - 50 micrometersPixel3 – 24 um Affymetrix Tiling Strategies• Standard• Alternative•Block• ExpressionAffymetrix Standard Tiling•Purpose: detection of mutations and polymorphisms and determination of which base is at a certain position.•Probes are arranged in sets of 4.•Each probe in a set of 4 has one of 4 bases at the substitution position.•Compares the four target-to-probe hybrid intensities in each set to identify the base in the substitution position.Affymetrix Standard TilingC-T-C-C-A-A-A-A-A-A-A-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-C-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-G-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-T-T-T-T-C-A-T-T-C-TSubstitution position5Affymetrix AlternativeTiling•Purpose: determination of base where multiple mutations are close together as opposed to a single point mutation.•Probes are arranged in sets of 5.•Includes a single base deletion at substitution point.•Compares the four target-to-probe hybrid intensities in each set to identify the base in the substitution position.Affymetrix Alternative TilingSubstitution positionC-T-C-C-A-A-A-A-A-A-A-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-C-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-G-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-T-T-T-T-C-A-T-T-C-TC-T-C-C-A-A-A-A-A-A- -T-T-T-C-A-T-T-C-TAffymetrix Block Tiling•Purpose: determination of genotype – wildtype or mutant. Determines which alleleis present.•Probes are arranged in sets of 5.•Includes a single base deletion at substitution point.•Compares the four target-to-probe hybrid intensities in each set to identify the base in the substitution position.Affymetrix Block TilingSubstitution positionC-T-C-C-A-A-A-A-A-A-A-A-C-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-C-A-C-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-G-A-C-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-T-A-C-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-A-A-G-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-C-A-G-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-G-A-G-A-G-A-T-T-C-TC-T-C-C-A-A-A-A-A-A-T-A-G-A-G-A-T-T-C-T-2 -1MS+1 +2Affymetrix Expression Tiling•Purpose: measure the relative abundance of various mRNA’s.•A set of probe pairs for each mRNA•PM – perfect match•MM – mismatch by one base•Software compares the hybridization intensities of the PM to those of the MM to determine the absolute or difference call for each probe set.Affymetrix Expression TilingPMACGGATGMM ACAGATGTARGETACGGATG6Data Analysis for Gene Expression*.cel file (pixel readings)*.dat file (average intensities, etc. are calculated)*.chp file (parameters are calculated)data mining, statistical analysisRaw Data to Cooked DataProbe Cell# of pixelsIntensityProbe cell Avg IntensityRaw Data to Cooked Data1. Calculate the Average Intensity of every probe cell2. Calculate the background3. Subtract the background4. Calculate the Noise (pixel-to-pixel variation within a probe cell)5. Determine numbers of Positive and Negative probe pairsfor every probe set.6. Positive Probe Pair = PM intensity > MM intensity7. Negative Probe Pair = MM intensity > PM intensity8. Calculate