Hi dear all, I am using limma for two color Agilent Microarray analysis. I found that normalizeBetweenArrays does not perform Quantile normalization directly to the M values. For that reason i had to use the following command MA_Quantile<- noralizeBetweenArrays(*MA$M*,method="quantile") To normalize M values i had to give MA$M instead of MA alone. I want to make sure whether this way it is correct or not and why M values are not normalized directly while it is mentioned in limma user guide that quantile method normalize M values. This question was raised earlier in Bioconductor list ( https://stat.ethz.ch/pipermail/bioconductor/2011-May/039618.html) but there is no answer for that. Thanks -- Neeraj KumarMolecular Reproduction Development and Genetics Prof. Kondaiah' Lab Indian Institute of Science Bangalore 560012 Mobile # +91-9986295625 [[alternative HTML version deleted]]

Hi Neeraj, One possible reason for the lack of responses is that you don't appear to have closely read the documentation for this method. >From ?normalizeBetweenArrays: Quantile normalization was explored by Yang and Thorne (2003) for two-color cDNA arrays. ?method="quantile"? ensures that the intensities have the same empirical distribution across arrays and across channels. ?method="Aquantile"? ensures that the A-values (average intensities) have the same empirical distribution across arrays leaving the M-values (log-ratios) unchanged. These two methods are called "q" and "Aq" respectively in Yang and Thorne (2003). I don't see anything there that would indicate that the M-values are quantile normalized, do you? It instead appears that the individual channels are quantile normalized, unless you specify Aquantile, in which the geometric mean intensities are normalized. Now if we look at the limma User's Guide, under section 6.3, we have: "This section explores some of the methods available for between-array normalization of two-color arrays. A feature which distinguishes most of these methods from within-array normalization is the focus on the individual red and green intensity values rather than merely on the log-ratios. These methods might therefore be called individual channel or separate channel normalization methods." And a bit later we have: "Applying quantile normalization directly to the individual red and green intensities produces a similar result but is somewhat noisier: > MA.q <- normalizeBetweenArrays(RG.b, method="quantile")" So in at least three instances it is clearly specified that quantile normalization for two-color arrays is not performed on the M-values, and a reference to the rationale behind this process is also provided. Best, Jim On Friday, September 13, 2013 1:17:51 AM, Neeraj Rana wrote: > Hi dear all, > I am using limma for two color Agilent Microarray analysis. > I found that normalizeBetweenArrays does not perform Quantile normalization > directly to the M values. > For that reason i had to use the following command > MA_Quantile<- noralizeBetweenArrays(*MA$M*,method="quantile") > To normalize M values i had to give MA$M instead of MA alone. > I want to make sure whether this way it is correct or not and why M values > are not normalized directly while it is mentioned in limma user guide that > quantile method normalize M values. > > This question was raised earlier in Bioconductor list ( > https://stat.ethz.ch/pipermail/bioconductor/2011-May/039618.html) but there > is no answer for that. > > Thanks > > > -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099