Dear BioConductor mailing list!
I perform 2 color hybridization with Exiqon slides, where each mir has 4
replicates. I compare a rather small number of slides (from 2 up to 6
biologial replicates for each treatment).
For analysis of the data I am using the R-2.8, the limma package and its
interface limmaGUI.
 That's what I do:
loading Genepix files
background correction (normexp, cutoff=10),
setting the GenePix Weightings: unflagged= 1, everything else= 0,
within (global loess) and between (Scale) array normalization.
To obtain the statistics for differential expression I choose the "least
squares" linear model fit and the calculation of Duplicate correlation.
Seems to work well, the problem is, that not always all 4 replicates of one
mir are "unflagged". It can happen that on the same slide only 2 or 3
replicates are unflagged, or that on one slide all of the 4 replicates are
flagged bad, but on the other slides all replicates for the same mir are
unflagged.
If I understood well, setting the weights only means that the intensities
from spots with zero weight do not influence the normalization.
How do I exclude the spots with zero weigths from the further calculations
of the linear model fit, is there a way in limmaGUI to exlude the bad spots
from the toptable list?
Thanks a lot in advance.
Greetings, Christine



Dr. Christine Völlenkle, Ph.D.
Research Laboratories-Molecular Cardiology
I.R.C.C.S. Policlinico San Donato
Via R. Morandi, 30
20097  S. Donato M.se (MI) Italy
Phone: +39 02 52774 683 (lab)
           +39 02 52774 533 (office)
Fax:    +39 02 52774 666
email: christine.voellenkle@gmail.com

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