Hi I have a small array that contains lots of spots for genes that *should* show no differential expression, and guess what? They don't! The raw data for these spots looks great, they all appear in a really tight cluster on an M vs A plot and have a log2 ratio of just above zero, which can easily be explained by dye bias. HOWEVER, the general trend on the M vs A plot, when a loess line is fitted, is not so good. In fact, M shows a negative relationship with A. Therefore, when I loess normalise, it pushes all my log2 ratios for the house-keeping genes up to around 1! I'm at a bit of a loss as to what to do - if I assume my house-keeping genes should all be zero, I will push the trend of M decreasing with A even further down. If I correct for this trend using loess normalisation, my house keeping genes will all display a two-fold up-regulation.... :-( Hmmmm. Mick

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