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Data Analysis DNA Microarrays. Dr. Rebecca Fry. Raw Data File. ID Cy3 Spot Mean Cy3 Background Cy5 mean Cy5 Background. Data Analysis. Step 1-Subtract Background Intensity from each spot. Generate Excel Formula. Paste into all cells. Data Analysis.
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Data AnalysisDNA Microarrays Dr. Rebecca Fry
Raw Data File ID Cy3 Spot Mean Cy3 Background Cy5 mean Cy5 Background
Data Analysis • Step 1-Subtract Background Intensity from each spot
Data Analysis • Step 2-Caculate average cy3 and cy5 intensities for control spots to determine normalization factor
Calculate average signals for cy3 and cy5 cy3=32 cy5=9 cy3/cy5= 3.55
Normalization factor= cy3/cy5=# #=normalization factor Our data=3.55
Data Analysis • Step 3-Normalize data using cy5 adjustment factor
Data Analysis • Step 4-Filter Genes showing no expression in both channels • Filter Genes with ½ intensity (backgound subtracted, normalized) of the average control intensity in both channels
Data Analysis • Step 5-Calculate Ratios of Gene expression change between final intensity value (background subtracted, normalized) Oligofectamine control (cy3) and knockdown (cy5) • Cy3/cy5=ratio=fold change
Data Analysis • Step 6-Generate Summaries • Number of Genes with differential gene expression fold change above a cutoff (1.5) • Number of Genes expressed on array (need not be differential) • How do you explain number of genes expressed (biology/technology)? • How could you test this hypothesis?