[BioC] presence/absence call using oligo array?

[xpeng]

Hi Amy,

Thanks for the reply.

You are right. The uninfected tissue is a blank, in theory. But the real 
problem is that some probes cross-hybridize with house RNAs. I do not know the 
oligo sequence yet. They know that there are more host RNAs than parasite RNAs 
in the sample from the infected tissue, but not sure the percentage.

Yes, they are 2-color arrays. The problem here is what to compare. It was 
suggested to find genes with intensities higher than their own backgrounds, 
say 4-fold higer, but I doubt it. I appreciate more inputs on the consequences 
if it is done this way.

Best,
Xinxia
>===== Original Message From a.mikhail at abdn.ac.uk =====
>Hi Xinxia,
>
>Just curious about your message.  It is quite common to look at parasite
>genes being expressed at different stages of the parasite lifecycle,
>including when the parasite is in the host, as is the case for your
>infected tissue.  However I'm a bit confused about the RNA you
>cohybridised with, as if there are no parasite RNAs in it you would be
>comparing parasite gene expression in your infected tissue with a blank
>(did I read correctly that your microarrays have only parasite genes on
>them, not host genes?).
>
>Are you using 2-colour arrays?  If so expression levels for each gene are
>normally read from one condition relative to the other.  This means that
>whatever you co-hybridised with has to have the same set of genes being
>expressed at some level - otherwise you have nothing to compare your
>condition of interest with.  The results you get in this way are relative
>expression levels for each gene of interest, not absolute expression
>levels. As I understand it you cannot use microarrays for absolute
>expression levels because there are too many factors that can disrupt mRNA
>copy number during probe preparation - if you are interested in actual
>copy number you would have to do something like quantitative RT-PCR.
>
>Hope this helps,
>
>Regards,
>Amy
>
>----------------------------------------------------------------------------
>Dear Bioc,
>
>Has someone ever used long oligo arrays to test if genes were expressed or
>
>not? I have two microarray slides on which oligonucleotides (65mer) for genes
>from a parasite genome were spotted. One gene has one oligo but spotted twice
>on each array. One slide has a RNA sample extracted from infected tissues
>(therefore mixture of both parasite RNAs and RNAs from host tissues)
>co-hybridized with RNAs from uninfected host tissue (no parasite RNAs).
>Another is a biological replicate but with dye-swap. People want to know
>what
>parasite genes are expressed in the infected tissue. Is it possible to do so?
>Related publications are highly appreciated.
>
>Best,
>Xinxia
>----------------------------------------------------------------------------
>
>
>
>-------------------------------------------
>Amy Mikhail
>Research student
>University of Aberdeen
>Zoology Building
>Tillydrone Avenue
>Aberdeen AB24 2TZ
>Scotland
>Email: a.mikhail at abdn.ac.uk
>Phone: 00-44-1224-272880 (lab)