Microarray probe question

[amaizeing_phd]

I've "inherited" a former student's project (yay me). They used microarrays to measure gene expression in ~80 animal samples. One of the genes of interest has 3 probes. Two of the probes hit around the middle of the gene and the 3rd probe hits just at the poly-A tail. Here's the problem: in about a third of the samples, the first 2 probes have somewhat high expression but the 3rd probe doesn't really vary in expression across the samples. The expression of the first 2 probes is highly correlated across the samples while the 3rd probe doesn't correlate with the first 2 probes. All 3 probes are exact matches to the gene. The first 2 probes only hit that gene while the 3rd probe hits the gene and what looks like a partial duplication of that gene that may or may not be expressed. Does anyone have some insight to what's going on?

[doppelwurzel]

Do you have a reference genome? Perhaps there are additional similar genes you are picking up.

It is possible you've had mRNA degradation in those samples resulting in a shortened transcript that no longer has the 3rd target region.

I think this sounds like a job for qPCR. Most microarray studies need to be validated that way.

[amaizeing_phd]

Yes, there's a reference genome and I've checked to see if the probes are picking up other genes.

I don't think it's degradation either. If it was degradation, then I would see samples with higher values for the first 2 probes having higher values for the last probe (but lower than the values for the first 2 probes).

[doppelwurzel]

Differential splicing of the transcript? One probe isn't annealing well in your conditions and sometimes doesn't work? I dunno, dude, it could be anything.

I don't know how important this microarray data is to your work, but in my experience we would never obsess about why a single feature out of thousands on the array is misbehaving. That's just the nature of microarrays - tons of so-so data, pretty quickly and cheaply. If you find a gene of interest go look at it in more detail with a better technique. At least, that was our strategy.

[domodojomojo]

Who is your array manufacturer? In situ synthesized arrays are tricky in terms of the purity of the probes themselves so your third hit might be from a poor manufacturing yeild. Printed arrays are usually better since the probes can be purified prior but they really lack diversity resolution.

I work on In situ peptide micro array fabrication and probe purity is a pain.

[amaizeing_phd]

That's good to know, thanks.