Dear BioC list,
I am working with Exiqon miRNA arrays, performing 2-color labeling,
comparing a cell line control versus cel line treatment
I am trying to find a method to control for labeling bias. I read postings
concerning this topic in the BioC archives, it says that the M values should
be negatively correlated. If they are not, I found the advice to go back to
the R and G intensities and check if they show the exepcted strongly
negative correlation.
I did both and saw the following:
R vs R as well as G vs G strongly positive: 0.94 (not normalized)
 M vs M  negatively correlated (-0.344 not normalized; -0.46 loess
normalized).

I do not understand how this is possible, did I do a mess in the script? If
there is a problem with dye degradation, I should see it also in the M vs M
correlation  as a positve correlation.
Is there any another way to check for labeling bias?
Thanks in advance for your time!
Christine

Please find enclosed the script:
  library("limma")
short_RG<-read.maimages(file="C:/Christine/CV 67.gpr",
source="genepix.median",
other.columns="Flags")

 ds_short_RG<-read.maimages(file="C:/Christine/CV 67_DyeSwap.gpr",
source="genepix.median",
other.columns="Flags")
   x= short_RG$R
y= ds_short_RG$R
cor(x,y)
 #### correlation 0.942051
plot (x=x, y=y)

 x= short_RG$G
y= ds_short_RG$G
cor(x,y)
 #### correlation 0.9397048
plot (x=x, y=y)


 # calculating M values, WIthout normalization
short_RG_M <- normalizeWithinArrays(short_RG, method="none",bc.method =
"normexp", offset = 10)
ds_short_RG_M <- normalizeWithinArrays(ds_short_RG, method="none",bc.method
= "normexp", offset = 10)

 x= short_RG_M $M
y= ds_short_RG_M$M
cor(x,y)
 #### correlation  -0.3445266
plot (x=x, y=y)


  # one-stop normexp bg correction and loess normalization
RG_loessB <- normalizeWithinArrays(short_RG, method="loess", bc.method =
"normexp", offset = 10)
ds_RG_loessB <- normalizeWithinArrays(ds_short_RG, method="loess", bc.method
= "normexp", offset = 10)
 x= RG_loessB$M
y= ds_RG_loessB$M
cor(x,y)
 #### correlation    -0.4643207
plot (x=x, y=y)

-- 
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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