[BioC] Doubt/Help - Microarray in R 2.12.2

[Vinicius Ribeiro]

Hello,

My name is Vinicius Ribeiro and I’m a PhD student in University of São Paulo (Brazil). I would like to solve a doubt regarding my microarray experiment that I carried out. I used a comparison between wild type and mutant Listeria monocytogenes (bacteria) in stress conditions.

Below you can find the sum of the commands that I used in my analysis. I had some warning messages and in the end of the processes I can not see p adj value <0.05 even with a lot of green and red spots in my slides (cy3 and cy5). I perfomed a similar experiment but using slides with 4 replicates of each probe and got excelent results but now using this slide (2 reps) I can not analyse properly.

R commands

> library(limma)

> library(statmod)

> targets.J1andC6<-readTargets("Targets_VR_2012.txt")

> raw.J1andC6<-read.maimages(targets.J1andC6, source="genepix", wt.fun=wtflags(0))

Read Slide 75_913_0700.gpr   
Read Slide 76_913_0700.gpr   
Read Slide 83_835_0656.gpr   

>  sorted.raw.J1andC6<-raw.J1andC6[order(raw.J1andC6$genes[,"Name"]),]

> norm.J1andC6<-normalizeWithinArrays(sorted.raw.J1andC6, method="printtiploess",bc.method="none")

> plotMA3by2(norm.J1andC6)

> design=c(-1,-1,1)

> corfit<-duplicateCorrelation(norm.J1andC6,design,ndup=2,spacing=1)

> corfit$consensus

[1] 0.7557859

> fit=lmFit(norm.J1andC6,design,ndup=2,correlation=corfit$consensus)

> fit2<-eBayes(fit)

Warning Message:

Zero sample variances detected, have been offset 

> topTable(fit2,number=100,adjust="BH")

     Block Row Column         ID    Name      logFC  AveExpr         t

1210     8  15      5 LM00001775 lmo2097  1.0534570 12.33612  4.180630

1161     4  15      3 LM00002616 lmo1999  1.2559634 11.25120  4.019180

378      4   4     13 LM00002219 lmo0433  1.9634563 12.87347  3.340647

Continue...

          P.Value adj.P.Val         B

1210 0.0008789322         1 -4.286804

1161 0.0016090372         1 -4.328428

378  0.0047104593        1 -4.359437

Continue...

> write.fit(fit2,file="J1andC6fit.txt",adjust="BH")



I don´t know if this helps but attached you can find my raw data. I used GenePix to scan.

Could anyone help me please. Is there other analysis that I can try?

Thank you so much.

Vinicius
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