Reading Affymetrix data from pd.hugene.2.0.st using oligo for RNA degradation plot

Question

[Vasu]

Hi,

I have some Affymetrix CEL files that use the HuGene 2.0 ST design.

I'm using oligo package to read CEL files. My question is how can I get RNA degradation plot using oligo?

Thanks

Answer 1

[Charles Warden]

You can get probe sequences and coordinates (usually) from the Affy website:

You'll want to extract the probe-level summarization and you can then plot intensity as a function of position with your genes of interest.

With respect to the probe-level summarization from the oligo package, the code should look something like this:

library(oligo)

celFiles = list.celfiles("/path/to/folder", full.names=T)
rawData = read.celfiles(celFiles)
probesetData = rma(rawData, target="probeset")
probeset.expression = exprs(probesetData)
probesets = rownames(probesetData)

However, selecting the optimal genes may be tricky. I've tried using the validated genes from this paper, but they didn't seem to show much of a position degradation effect (in the array data that I have checked so far):

[Vasu]

Hi Charles,

Thanks for the reply. I have used "xps" package for this.

And I got the following plot. Help Needed to Interpret RNA degradation plot.

Thank you in Advance

[Charles Warden]

I'm not exactly sure how to interpret the figure - I would have expected more of a 5' degradation bias. At least the intensities are relatively consistent within each gene, so I would say the signal isn't too noisy.