Hello to everyone,

I am trying to find gene fold change for transcription data in R via limma package.As I am bit new to this, just want to make sure that what I have been doing is correct or no. I am bit more concern on design matrix that i have created correct or not and also with the contrast matrix.  My target is to get relative gene fold change from control vs treatment at 3 time points and the transcription data (microarray data) are like this; I have two control replicates at each time point of 2hr, 8hr and 24hr. The treatment groups includes 3 doses lower, middle and higher with each two replicates at time point of 2hr, 8hr and 24hr. So total I have 24 samples including both control, treatment. I want to compare the control vs treatment at each time point like

Here below I have attached the code, please check it.

samples <- paste(targets$Target,  targets$time ,sep="/")

sample<-  factor(samples)

design <- model.matrix(~0+sample)

colnames(design) <- levels(sample)

 Control2/2 hr Control24/24 hr Control8/8 hr High2/2 hr High24/24 hr High8/8 hr

1              1               0             0          0            0          0
2              1               0             0          0            0          0
3              0               0             0          0            0          0
4              0               0             0          0            0          0
5              0               0             0          0            0          0
6              0               0             0          0            0          0
7              0               0             0          1            0          0
8              0               0             0          1            0          0
9              0               0             1          0            0          0
10             0               0             1          0            0          0
11             0               0             0          0            0          0
12             0               0             0          0            0          0
13             0               0             0          0            0          0
14             0               0             0          0            0          0
15             0               0             0          0            0          1
16             0               0             0          0            0          1
17             0               1             0          0            0          0
18             0               1             0          0            0          0
19             0               0             0          0            0          0
20             0               0             0          0            0          0
21             0               0             0          0            0          0
22             0               0             0          0            0          0
23             0               0             0          0            1          0
24             0               0             0          0            1          0
   Low2/2 hr Low24/24 hr Low8/8 hr Middl8/8 hr Middle2/2 hr Middle24/24 hr
1          0           0         0           0            0              0
2          0           0         0           0            0              0
3          1           0         0           0            0              0
4          1           0         0           0            0              0
5          0           0         0           0            1              0
6          0           0         0           0            1              0
7          0           0         0           0            0              0
8          0           0         0           0            0              0
9          0           0         0           0            0              0
10         0           0         0           0            0              0
11         0           0         1           0            0              0
12         0           0         1           0            0              0
13         0           0         0           1            0              0
14         0           0         0           1            0              0
15         0           0         0           0            0              0
16         0           0         0           0            0              0
17         0           0         0           0            0              0
18         0           0         0           0            0              0
19         0           1         0           0            0              0
20         0           1         0           0            0              0
21         0           0         0           0            0              1
22         0           0         0           0            0              1
23         0           0         0           0            0              0
24         0           0         0           0            0              0
attr(,"assign")
[1] 1 1 1 1 1 1 1 1 1 1 1 1
attr(,"contrasts")
attr(,"contrasts")$f
[1] "contr.treatment"

contrast.matrix <- makeContrasts(control_lowdose.2 = Low2-Control2, control_lowdose.8 = Low8 -       Control8,

                                 control_lowdose.24 = Low24-Control24,control_Mediumdose.2 = Middle2 -Control2,

                                 control_Mediumdose.8 = Middl8 -Control8, control_Mediumdose.24 = Middle24-Control24 ,

                                 control_Highdose.2 = High2 -Control2,control_Highdose.8 = High8 -Control8,

                                 control_Highdose.24 = High24-Control24,levels=design)

The names in your makeContrasts call do not match up with the column names of design. The latter also contain some characters (e.g., space, forward slash) that make them incompatible with makeContrasts.

Other than that, the overall parametrization of your experimental design looks fine.