Hi again,

I have an RNA-seq experiment with samples of different genotypes and 3 different developmental stages. For each sample I determined a "genostage" factor

genotype <- rep(c("WT","Geno1","Geno2",<other genotypes>), each = 12)
dev <- rep(c("stage1","stage2", <other stages>), each = 4)
genostage <- factor(paste(genotype, dev, sep = "_"))
design <- model.matrix(~0+genostage)

Now, I would like to define the contrast to determine changes in expression over time (developmental stage) between two genotypes. According to examples in the limma manual, I would define the contrast like that:

(genostageG1_stage2-genostageG1_stage1)-(genostageG2_stage2-genostageG2_stage1)

But then, I was wondering how I would interpret the results of the following contrast, and whether it makes sense at all?

(genostageG1_stage2-genostageG2_stage2)-(genostageG1_stage1-genostageG2_stage1)

Could you please shortly explain me the difference between these two contrast statements?

Thank you very much.

There is no difference between these two contrasts. One is just an arithmetic rearrangement of the other.

Thanks for your response. If it is just an arithmetic rearrangement, shouldn't there be a "+" somewhere?

Irrespectively, my results show the same number of differentially expressed genes for both contrasts, so in total 203 genes show expression changes over development between the 2 genotypes.

But, how do I interpret the "1" and "-1" genes in each contrast?

For case 1: I would say, that 90 genes are preferentially regulated within G1 between the two developmental stages, and 113 genes are preferentially regulated in G2 between the two developmental stages.

For case 2: I would say, that 113 genes are preferentially regulated within developmental stage 2 between G1 and G2, and 90 genes are preferentially regulated within developmental stage 1 between G1 and G2.

Aren't these two totally different statements?

(genostageG1_stage2-genostageG1_stage1)-(genostageG2_stage2-genostageG2_stage1)
-1                                      113
0                                     28390
1                                        90
(genostageG1_stage2-genostageG2_stage2)-(genostageG1_stage1-genostageG2_stage1)
-1                                      90
0                                    28390
1                                      113