Dear Limma users,
   
  Although I have read the manual and searched the mailing list, I still do not fully understandf how weights are used when applying the linear model.
   
  In our case we have four cDNA microarrays (2 biological replicates with 2 dye-swaps for each) in which we compare a mutant with a wt sample. The microarrays have each gene printed once. We use different spot quality filters and we apply a 0.01 weight to the spots which do not meet these criteria or which are controls. We then apply lmFit with weights and use BH to adjust for multiple testing. These downweighted spots do not affect the Loess print-tip normalization, but how are they treated in the linear model?
   
  For example, some of the genes with 3 replicate spots with weight 0.01 and 1 with weight 1 have an adjusted P value < 0.05. Is Limma "taking into consideration" that 3 of the spots are suboptimal when calculating the Adj. P.Vals.? Could this gene be considered fairly reliable because although 3 of the spots are suboptimal the fact that they have a similar ratio than the one which is optimal "adds strength" to the differential expression of that gene?
   
  If we set the 0.01 weights to 0.00 after the normalization, Adj.P Values are not calculated (as expected displays NA) for those genes with all four weights being 0. However, P values are calculated for those genes with only one weight being 1. How come  there is a Pval in the latter if there is only 1 optimal observation?
   
  The M values are almost identical doing it this way or leaving the weights at 0.01, but the Adj.P.Vals are somewhat different (Example: 3000 genes with P<0.05 doing it either way but around 1000 genes are < 0.05 only by doing it one way or the other). However, no differences with genes over log2ratio 1 or -1 and very slight ones (12 genes)  with genes log2ratio < 0.58 or -0.58.
   
  In summary, after obtaining the Limma results list, would you set an extra filter to select only those genes which have N spots meeting the quality criteria? Is it necessary?  Which value should N be? Thank you very much,
   
  A. Juncal

 		
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