1、Microarrays for Gene Expression AnalysisQuestions: What genes are expressed in this tissue under these conditions?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 tis

2、sue?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 Gen

3、esUnderstanding Gene Regulatory NetworksExpressed Genes = mRNADNA messenger RNA protein Expressed Genes = Currently TranscribedmRNAExtract RNAmRNAmRNAmRNAmRNAIsolate mRNAsAffymetrix Oriented Fluorescently tagged cRNA One chip per sample One for control One for each experiment Other methods include t

4、wo dyes/one chip Red dye Green dye Control and experiment on same chipCreating TargetsmRNAReverse TranscriptasecDNAPCR Amplificationof DNAIn Vitro transcription to create cRNARNA-DNA Hybridizationprobe setsDNA(25 base oligonucleotides of known sequence)TargetsRNANon-Hybridized Targets are Washed Awa

5、y“probe sets (oligos)Targets(fluorescently tagged)Non-bound ones are washed awayPicture of Gene ChipHandling ChipArgon laser488nm 570nm Scanner based on epifluorescence confocal microscopy Custom Chips vs Affy Chips Affy chips contains thousands of gene probes Genes selected from sources such as Gen

6、Bank 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 CYP450s, Heat Shock proteins Drug transporters Stress-activated kinases Rat Neurobiology chip - 1,200 genes Synuclein 1, prion

7、protein, Huntingtons disease Syntaxin, Neurexin, neurotransmitters Example Affy Chips Arabidopsis Genome Chip Murine Genome Chip - 36,000 genes E. coli Genome Chip - 4,200 ORFs Drosophila Genome Chip - 13,500 sequences Yeast Genome Chip - 6,400 ORFs Human Genome Chip - 60,000 human genes Definitions

8、Probe a single-stranded DNA oligonucleotide complementary 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 an

9、d 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 sequen

10、ce 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 MM intensityNegative Probe Pair = MM intensity PM intensityCalculate Positive FractionCalculate Pos/Neg RatioCalculate

11、Log Average Ratio & Avg DifferenceAbsolute Analysis Parameters Probe Set Name Positive - number of pairs scored positive Negative number of pairs scored negative Pairs number of probe pairs for a probe set Pairs Used those not masked for some reason PairsInAvg excludes those with extremely intense o

12、r weak scores informationalQuality controlAbsolute Analysis Parameters PM Excess have exceeded limit for intensity MM Excess have exceeded limit for intensity Avg Diff average difference of fluorescence intensity between the PM and MM cells. Log Avg Ratio a measure of the hybridization performance H

13、igher = better Log Avg = 0 indicates random cross hybridization Pos/Neg ratio of positive probe pairs to negative probe pairs Positive Fraction positive probe pairs/probe pairs Abs Call Present, Absent or Marginal. Is this gene present in this sample?Quality controlRaw Data to Cooked DataPositive Fr

14、action Pos/Neg Ratio Log Avg RatioDecision MatrixAbsolute Call(Present, Absent, Marginal) Data AnalysisAbsolute Analysis used to determine whether transcripts represented on the probe array are detected or not within one sample(uses data from one probe array experiment).Comparison Analysis used to d

15、etermine the relative change in abundance for each transcript between a baseline and an experimental sample(uses data from two probe array experiments). Intensities for each experiment are compared to a baseline/control.Approaches What genes are Present/Absent in my tissue? What genes are Present/Ab

16、sent in the experiment vs control? Which genes have increased/decreased expression in experiment vs control? Which genes have biological significance based on my knowledge of the biological system under investigation?Approaches to Data AnalysisDatabase QueriesGraphical AnalysisStatistical AnalysisBi

17、ological KnowledgeSet Filter ParametersQueryPivotScatter/Fold GraphSelect PointsIdentify interestingrelationshipsAdd probe sets to filterBar GraphAdjust filter parametersData AnalysisAbsolute Analysis used to determine whether transcripts represented on the probe array are detected or not within one

18、 sample(uses data from one probe array experiment).Comparison Analysis used to determine the relative change in abundance for each transcript between a baseline and an experimental sample(uses data from two probe array experiments). Intensities for each experiment are compared to a baseline/control.

19、Comparison Analysis Parameters Inc number of probe pairs that increasedDec number of probe pairs that decreased Inc Ratio Dec Ratio Max Inc & Dec Ratio Pos Change Neg Change Inc/Dec DPos-DNeg Ratio Log Avg Ratio Change Diff Call did this gene increase or decrease? Increase, Marginal Increase, Decrea

20、se, Marginal Decrease, No Change Avg Diff Change how much did the difference between PM and MM change from the control to the treated?(Avg Dif Exp Avg Dif Control) B=A was this gene present in the control? Fold Change how many times more expression did the treated have compared to the control? (posi

21、tive or negative) Sort Score a ranking based fold change and avg diff changeComparison Analysis Parameters(continued)Data AnalysisFilter/Query:Select those oligos which have shown a real,significant change.Filter & Sort to Find Real ChangesAvg. Difference Change = 200 andFoldChange 3 andINC = 70% an

22、d DEC = 0%Query ResultsQuery ResultsProbe1Probe2Probe3Exp1 1 4 9Exp2 3 6 8Pivot the Query Results Experiments = columns Genes = Rows Shows how genes change across experimentsPivot ResultsExp1Exp2Probe1 1 3Probe2 4 6Probe3 9 8Pivoted DataCan be sorted by any parameter. Sort in descending order to sho

23、w greatest differences.Description of geneLink to NCBIGraphical AnalysesScatter Plot Graph requires Control vs ExperimentHow many times did the expression of this gene change in the treated tissue versus the control?Fold Change GraphsStatistical TechniquesStatistical TechniquesSelf-Organizing MapsCo

24、rrelation Coefficient ClusteringAnalysis FunctionMatrixSelf-Organizing MapsClusteringAutomatically discovering classesSelf-Organizing MapsSelf-Organizing MapsGenes whose expression level rise/fall togetherunder the same conditions, cluster togetherCo-regulated ?Statistical Techniques Average Standard Devia