Hi:

I am experimenting preprocessed Affymetrix microarrays expression data matrix (Affymetrix probe-sets in rows (32830 probesets), and RNA samples in columns (735 samples)) for my downstream analysis. However, I have `pheno`

metadata for the experiment observations.

**data that I used**:

```
> dim(eset_HTA20)
[1] 32830 735
> eset_HTA20[1:3, 1:3]
Tarca_001_P1A01 Tarca_003_P1A03 Tarca_004_P1A04
1_at 6.062215 6.125023 5.875502
10_at 3.796484 3.805305 3.450245
100_at 5.849338 6.191562 6.550525
> dim(phenoDat)
[1] 735 7
> phenoDat[1:3,]
SampleID GA Batch Set Train Platform
Tarca_001_P1A01 Tarca_001_P1A01 11.0 1 PRB_HTA 1 HTA20
Tarca_003_P1A03 Tarca_013_P1B01 15.3 1 PRB_HTA 1 HTA20
Tarca_004_P1A04 Tarca_025_P1C01 21.7 1 PRB_HTA 1 HTA20
T
Tarca_001_P1A01 Preterm
Tarca_003_P1A03 Preterm
Tarca_004_P1A04 Preterm
```

**my attempt**:

I tried differential expression analysis on `eset_HTA20`

data, here is my workflow by using `limma`

package:

```
ano <- phenoDat
esetHTA <- HTA20_rma_filt
ano$ID <- factor(ano$SampleID)
designs <- model.matrix(~0+T+SampleID, ano)
esets <- eset_HTA20[, rownames(ano)]
colnames(designs)<-substr(colnames(designs),2,100)
fit <- lmFit(esets, designs)
cont.matrix <- makeContrasts( contrasts="Term-Preterm",levels=designs)
fit2 <- contrasts.fit(fit, cont.matrix)
fit2 <- eBayes(fit2)
```

but when I ran `fit2 <- eBayes(fit2)`

, I got the following error:

```
> fit2 <- eBayes(fit2)
Error in .ebayes(fit = fit, proportion = proportion, stdev.coef.lim = stdev.coef.lim, :
No residual degrees of freedom in linear model fits
```

I looked into support site here and Gordon explained several threads that this happened if data doesn't have replicate. I am not quite sure how to proceed with my analysis now. How can I know data have replicates or not? If there is not replicate, what's the possible solution to go through with this issue? can anyone point me out any possible strategy I could try here? any thought?

bingo, I just fixed this by taking your opinion, but why I should include

`Batch`

instead of`SampleID`

? I am a newbie for differential expression analysis, so this is not crystal clear to me yet. Could you give extended comments on your proposal? I want to learn? what else I have to try for`designs`

matrix? any idea?To be blunt, this is not a set to use learn on. It's way too large and complex. If you don't understand why batch should be a category and sample ID should not, you need to practice with tutorials and much smaller sets so that you understand the basics of what you are doing.