Hi I have an experiment with a reference design. My reference is a good one, in that it lights up every spot brightly, is constant and there is lots of it. The result of this, when I look at an M vs A plot, is that there is a high positive correlation between M and A - as my reference (Cy3) remains pretty much constant, then as Cy5 increases, so do both M (log(ratio)) and A(average log(intensity)) (this can be demonstrated by plotting M vs log(Cy5) and M vs log(Cy3) - the positive correlation between M and A definitely comes from the Cy5 values) This is presumably the correct shape for an M vs A plot for a reference design? So is it now right to perform loess normalisation? I expect the positive correlation between M and A, so is it right to remove it? If not, then what other normalisation method should I use given that I want to compare across several arrays all with the same reference? Thanks Mick