1Data AnalysisDNA MicroarraysDr. Rebecca FryRaw Data FileIDCy3 Spot MeanCy3 BackgroundCy5 meanCy5 Background2Data Analysisz Step 1-Subtract Background Intensity from each spotGenerate Excel Formula3Paste into all cells4Data Analysisz Step 2-Caculate average cy3 and cy5 intensities for control spots to determine normalization factorSort File Based on Clone ID to locate controls5File is now alphabetizedCalculate average signals for cy3 and cy56cy3=32cy5=9cy3/cy5=3.55Calculate average signals for cy3 and cy5z Normalization factor=cy3/cy5=##=normalization factorOur data=3.557Data Analysisz Step 3-Normalize data using cy5 adjustment factor8Paste into all cellsData Analysisz Step 4-Filter Genes showing no expression in both channelsz Filter Genes with ½ intensity (backgoundsubtracted, normalized) of the average control intensity in both channels9Generate Excel FormulaPaste into all cells10Filter genes that are not present in cy3 or cy5Data Analysisz Step 5-Calculate Ratios of Gene expression change between final intensity value (background subtracted, normalized) Oligofectamine control (cy3) and knockdown (cy5)z Cy3/cy5=ratio=fold change1112Why we filter!Data Analysisz Step 6-Generate Summariesz Number of Genes with differential gene expression fold change above a cutoff (1.5)z Number of Genes expressed on array (need not be differential)z How do you explain number of genes expressed (biology/technology)?z How could you test this