Question: Measuring Microrna Expression On Affymetrix Gene St Chips
6
gravatar for David Quigley
8.1 years ago by
David Quigley11k
San Francisco
David Quigley11k wrote:

We have recently started using the Affymetrix Human Gene ST 1.1 and Mouse Gene 1.1 ST arrays. These arrays are designed primarily to measure expression of well-annotated genes, not microRNAs. However, as of annotation revision 31, the Mouse Gene ST chip lists 341 microRNA probesets. For example, probeset 10409709 is annotated as Mir7-1, with 28 probes covering the transcript. Presumably these probes measure the full-length transcripts before processing.

Has anyone actually used these probesets to look at microRNA expression? Specific questions:

  1. Normalization: Did you just use gcRMA? We've some experience using ABI assays to measure microRNA expression, and the normalization methods are quite different there.
  2. Results: How do the results compare results to custom-designed assays? Do the probes work?

Anyone who has seen a reference to using these arrays for miRNA analysis in the literature, I'd be very grateful. We are considering changing our favorite RNA extraction protocol to take advantage of these probes, as normally we would discard microRNA-sized RNA. Is it worth the change?

NOTE: This question is distinct from the one recently asked. I know we can buy dedicated microarrays to measure microRNA levels; that's not what this question is about.

ADD COMMENTlink modified 8.1 years ago by Larry_Parnell16k • written 8.1 years ago by David Quigley11k

I didn't ask that question. Only tried to answer it.

ADD REPLYlink written 8.1 years ago by Chris Evelo9.9k

My apologies, I confused "most recent answer" with "asked question". Edited to reflect this.

ADD REPLYlink written 8.1 years ago by David Quigley11k

Funny enough, actually, I was absolutely thinking in that direction. Your question is more precise though ;)

ADD REPLYlink written 8.1 years ago by Doo240

I cannot answer your question, but what do think about this aspect: Ofc, if you are primarily interested in measuring exactly only those miRNAs represented on the chip it might work, fine. But isn't miRNA analysis mostly about finding new miRNAs and their targets? Is quantitation really so important in your case, because I think you might consider a miRNA-sequencing approach instead, if the costs are comparable.

ADD REPLYlink written 8.1 years ago by Michael Dondrup45k

I agree that if you want to look at miRNA, the proper way to do it is with a dedicated assay or sequencing approach. Some miRNAs have known (or at least published) activities as, say, tumor suppressors, and we know we would be interested in them. The main point is that we're already committed to using these chips; I just don't know if the "miRNAs that we get for free" would be trustworthy.

ADD REPLYlink written 8.1 years ago by David Quigley11k
1
gravatar for Larry_Parnell
8.1 years ago by
Larry_Parnell16k
Boston, MA USA
Larry_Parnell16k wrote:

David asked "We are considering changing our favorite RNA extraction protocol to take advantage of these probes, as normally we would discard microRNA-sized RNA. Is it worth the change?"

IMO, I would make the change but keep the RNA fractions separate, if possible. My concern is with smaller species that are not microRNAs influencing the larger-sized mRNA signals. Just a feeling - we have no experience with performing expression analysis as you outline in your question. We've bought those dedicated microRNA arrays.

ADD COMMENTlink written 8.1 years ago by Larry_Parnell16k
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