Analysis of microarray data - VSN International

34Analysis of Microarray Data in GenStatIn this experiment, which has only two treatments, we only need thedifference between the two treatments, and so this contrast of DM –Control is entered as -1 and 1 (right).The options menu can now be completed (right). The most importantoption in this menu is the one that specifies whether a dye bias isestimated from the dye swaps. If there are no dye swaps in theexperiment, this must be turned off, as otherwise the treatment estimateswill be confounded with the dye effect, and you will get an error in theoutput that will say that the design is disconnected. If you have balanceddye swaps in your experiment, the means will be the samewhether this is selected or not, but the variance of the dyeeffect will be removed from the residual variation, and onedegree of freedom will be taken of the residual degrees offreedom to account for this. In a reasonable sized experimentor in one not balanced for dyes, it is best to estimate the dyebias effect to get an unbiassed estimates of effects and theirstandard errors. In this experiment with only 4 slides, theoption to estimate dye bias will not be used to save a degree offreedom in the statistical tests, which decreases the size ofeffect needed to be significant at the risk of an effect beingupset by some dye bias.The Store button allows the results to be saved. Unlike theusual Save buttons on other GenStat menus, the columns to besaved must be specified before using the Run button. In theStore dialog shown to the right, every column possible hasbeen specified, and the option to display the results in aspreadsheet has been selected. Clicking the Run button willperform the analysis and produce the following spreadsheet.This spreadsheet contains a line for each probe, and columns for thedifferent targets and contrasts. The spreadsheet is in probe order, but canbe sorted into various orders using the Spread | Sort menu so that forexample all the most differentially expressed probes are at the start orend of the spreadsheet. The Sort dialog (right) shows the spreadsheetbeing sorted on the values of the Contrast (DM vs. C). The mostdifferentially under-expressed probes will be at the start of thespreadsheet, and those over-expressed will be at the end. Thisspreadsheet has been saved as “Estimates13-6-9.gsh”.