On 2/25/2013 8:14 AM, Fiona Ingleby wrote:
> Dear all,
> I am working with microRNA data and as it is very new to me, I have
some general questions. I'm hoping that some people on this list who
know more about microarray analysis and miRNA than myself might be
willing to offer some opinions/advice.
> First, when doing quality checks on the data, how useful is it to
look at the RNA degradation plots with miRNA, given how short miRNAs
Assuming here that you have an Affy miRNA array of some sort, not
at all. Note that the Affy probes are all 25-mers, and miRNA
are 21-23 nt long. In the vast majority of cases (with mature miRNA
transcripts; this doesn't apply to the scaRNA, snoRNA nor hp-miRNAs),
the duplicate probes are all identical. So the underlying premise
the RNA degradation plot doesn't hold.
> Second, when analysing differential expression (using lmFit and
eBayes in the 'limma' package), I find quite high numbers of probes
which are significantly differentially expressed across my samples (in
some contrasts as many as ~500 out of 4000 probes are significantly
differentially expressed between samples). Is this unusual with miRNA
data? Further, although a lot of these probes are from Drosophila
species (which I expected since my samples are from D. melanogaster),
some are from other insects, and even other phyla (there are some C.
elegans and human miRNA probes which are found to be significantly
differentially expressed, for example).
> From what I have read in the literature so far, there does seem to
be some conservation of miRNAs between diverse species, but I can't
find any information about to what extent I might expect to find this
in my data. I'm a little concerned that this level of homology between
species might be unusual, which would suggest that I've made a mistake
in the analysis.
> As I said, I am very new to this, so if anyone is willing to offer
advice or just point me in the direction of some useful information I
could look at myself, then I would really be very grateful.
In general I like to see somewhere around 15-30 differentially
miRNA transcripts, as anything more can become quite intractable for
people I collaborate with. This is because each miRNA may target >
mRNA species, so too many miRNAs and all of a sudden every gene might
a potential target.
That said, I just did an analysis looking at different brain regions
a particular bird species and there were hundreds to thousands of
differentially expressed miRNA transcripts, depending on the contrast.
So the fact that you have lots of differentially expressed transcripts
doesn't necessarily mean you made a mistake. In addition having probes
from other species pop up might not be a bad thing. In my experience,
you get the same miRNA from multiple species differentially expressed,
it is because the miRNA is highly conserved, and there are little or
differences in the sequence.
One thing we commonly do is to start with just those miRNA transcripts
that from the species under consideration. This can help limit things
a tractable number of miRNAs.
> Many thanks in advance,
> Dr Fiona C Ingleby
> Postdoctoral Research Fellow
> University of Sussex, UK
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James W. MacDonald, M.S.
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Environmental and Occupational Health Sciences
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