[BioC] Limma; Background and Single Channel Analysis

[Simone de Jong]

   Dear all,

   I  am  analyzing  data  from  44k agilent microarrays in limma. I have
   several issues where I'd like to hear some opinions from others.

   First,  I  have put the weights of the controlspots to 0 after reading
   in RG, in the command to normalize, is that ok?

   Second,  using  agilent  FE  flags,  not  recommended  I  read? Should
   anything  be  flagged  out beforehand, like signals <0? And what about
   the saturated ones?

   Third,  I  have  been experimenting with several background correction
   methods.  It turns out that 'normexp' gives good results for most, but
   not  all arrays. Two of them look really weird, with the MA cloud only
   starting  after  5.. I came to the conclusion that the method Subtract
   results  in the best looking MA plots for all of my arrays. The 'half'
   and 'minimum' method do not give such good results, with ugly stripes.
   But   now  (ofcourse  after  within  (loess) and  between  (aquantile)
   normalization),  I am not able to do 'single channel analysis' because
   the  intraspot  correlation  cannot process missing or infinite M or A
   values.. what to do?

   Fourth,  when  I  was experimenting with single channel analysis I was
   able  to  do a groupwise analysis, but the paired-samples analysis did
   not  work  because  of 'no residual degrees of freedom' error. We have
   hybridized  for  each control and each experimental subject 1 baseline
   sample  (-,  cy3)  and  1  challenged  (+,  cy5) sample. So we want to
   compare   the   +/-   ratio  for  each  experimental  subject  with  a
   specifically matched control, is that possible?

   Hope to read advise from you soon,
   Simone de Jong