thanks jim But when i use normalizeBetweenArrays(MA,methode="quamtile),it is supposed to perform quantile normalization on M values and the mean ,median,maximum value and minimum value should be the same of all arrays after normalization. I am copying my script here for reference. > targets <- readTargets("target.txt") > targets FileName Cy3 Cy5 1 207_1_1_4HR.txt 4HCY3 4HCY5 2 207_1_2_4HR.txt 4HCY3 4HCY5 3 207_1_3_1HR.txt 1HCY3 1HCY5 4 207_1_4_1HR.txt 1HCY3 1HCY5 5 8052_1_3_12HR.txt 12HCY3 12HCY5 6 8052_1_4_12HR.txt 12HCY3 12HCY5 > RG <- read.maimages(targets, source="agilent") Read 207_1_1_4HR.txt Read 207_1_2_4HR.txt Read 207_1_3_1HR.txt Read 207_1_4_1HR.txt Read 8052_1_3_12HR.txt Read 8052_1_4_12HR.txt > MA <- normalizeWithinArrays(RG,method="loess",bc.method="normexp") Array 1 corrected Array 2 corrected Array 3 corrected Array 4 corrected Array 5 corrected Array 6 corrected Array 1 corrected Array 2 corrected Array 3 corrected Array 4 corrected Array 5 corrected Array 6 corrected > *summary(MA$M)* 207_1_1_4HR 207_1_2_4HR 207_1_3_1HR 207_1_4_1HR 8052_1_3_12HR 8052_1_4_12HR Min. :-8.853926 Min. :-9.948604 Min. :-6.767666 Min. :-6.642965 Min. :-5.344051 Min. :-6.576922 1st Qu.:-0.395661 1st Qu.:-0.024118 1st Qu.:-0.364050 1st Qu.:-0.339771 1st Qu.:-0.308643 1st Qu.:-0.352430 Median :-0.002921 Median :-0.005917 Median :-0.007129 Median : 0.003566 Median : 0.007702 Median : 0.007684 Mean : 0.013726 Mean : 0.002334 Mean : 0.043019 Mean : 0.002412 Mean :-0.001696 Mean :-0.012679 3rd Qu.: 0.412789 3rd Qu.: 0.013281 3rd Qu.: 0.412566 3rd Qu.: 0.342954 3rd Qu.: 0.311236 3rd Qu.: 0.349261 Max. : 4.826212 Max. : 3.977841 Max. : 4.061991 Max. : 4.568052 Max. : 4.140642 Max. : 4.218073 > *MA <- normalizeBetweenArrays(MA,method="quantile")* >* summary(MA$M)* 207_1_1_4HR 207_1_2_4HR 207_1_3_1HR 207_1_4_1HR 8052_1_3_12HR 8052_1_4_12HR Min. :-6.921310 Min. :-13.368132 Min. :-5.118688 Min. :-6.108837 Min. :-4.488686 Min. :-5.388395 1st Qu.:-0.344347 1st Qu.: -0.238684 1st Qu.:-0.328855 1st Qu.:-0.316487 1st Qu.:-0.281210 1st Qu.:-0.344085 Median :-0.029286 Median : 0.009940 Median :-0.047600 Median : 0.000221 Median :-0.000253 Median :-0.002598 Mean :-0.000028 Mean : 0.000014 Mean :-0.000024 Mean :-0.000030 Mean : 0.000019 Mean : 0.000045 3rd Qu.: 0.312078 3rd Qu.: 0.244628 3rd Qu.: 0.287170 3rd Qu.: 0.315771 3rd Qu.: 0.273066 3rd Qu.: 0.341673 Max. : 5.689074 Max. : 5.190544 Max. : 5.034104 Max. : 5.185184 Max. : 4.800180 Max. : 5.212559 thanks On Wed, Sep 11, 2013 at 8:04 PM, James W. MacDonald <jmacdon@uw.edu> wrote: > Hi Neeraj, > > From ?normalizeBetweenArrays: > > object: a numeric ‘matrix’, ‘EListRaw’, ‘RGList’ or ‘MAList’ object > containing un-normalized expression data. ‘matrix’ or > ‘EListRaw’ objects will be assumed to contain single- channel > data, usually log-intensities, whereas ‘RGList’ or ‘MAList’ > objects should contain two-color data. > > > > On Wednesday, September 11, 2013 10:08:38 AM, Neeraj Rana wrote: > >> Dear Bioconductor List, >> >> I am doing two color Agilent microarray analysis by limma. >> >> I tried to normalize* M *values by using following command.But both the >> >> commands are giving different M values. >> >> *(1) MA<- normalizeBetweenArrays (MAw, method="quantile") >> > > So here limma thinks you have a two-color array. > > >> * >> *(2) MA <- **normalizeBetweenArrays (MAw$M, method="quantile") >> > > And here limma thinks you have a one-color array. Since you do not have a > one-color array, this is not what you should be doing. > > Best, > > Jim > > > > > >> * >> the first command does not give same boxplot of M value for all arrays , >> but second command is giving same boxplot for all arrays.* >> >> * >> *I want to know whether first command is proper for between the array >> >> normalization and is it applied on M values or not? >> >> * >> Thanks * >> * >> * >> ** >> * >> >> -- >> >> [[alternative HTML version deleted]] >> >> ______________________________**_________________ >> Bioconductor mailing list >> Bioconductor@r-project.org >> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat.e="" thz.ch="" mailman="" listinfo="" bioconductor=""> >> Search the archives: http://news.gmane.org/gmane.** >> science.biology.informatics.**conductor<http: news.gmane.org="" gmane="" .science.biology.informatics.conductor=""> >> > > -- > James W. MacDonald, M.S. > Biostatistician > University of Washington > Environmental and Occupational Health Sciences > 4225 Roosevelt Way NE, # 100 > Seattle WA 98105-6099 > -- Neeraj KumarMolecular Reproduction Development and Genetics Prof. Kondaiah' Lab Indian Institute of Science Bangalore 560012 Mobile # +91-9986295625 [[alternative HTML version deleted]]