Hi Igor,
I assume the array what you have used is a Nimblegen Expression array and you have performed two colour hybridization with a dye swap to address your question.. In order to estimate up-regulated genes in a given condition(condition is nothing but logCy5-logCy3) analyse the data with your dye swap (logCy3-logCy5) data in Limma. Theoretically the up regulated probes in the usual experiment should be down regulated in dye swap,however due to dye effect it will not be . Thats why limma and other software like Gene Spring performes Dye Swap Normalization and gives Upregulated and down regulated gene list. More details is present in the free access paper entitled as "Assessing the efficiency of dye-swap normalization to remove systematic bias from two-color microarray data". To present the data you can use Volcano Plot.
May I know how you calculated fold difference? As for as my understanding, fold difference is calculated in the following condition.....
Suppose you are trying to see efficacy of drug, then before treatment isolate mRNA and label with cy3 as well as cy5 and after treatment label mRNA with cy5. Do two hybridization one Cy3 of Untreated vs Cy5 of untreated, second Cy3 of Untreated vs Cy5 of Treated. So in this you have Cy3 Chanel is being same for both expt and you can estimate Fold change difference. But in your case, expt is performed some what - Cy3 untreated vs Cy5 treated with dye swap, where measuring fold change difference is not logically correct.
Thanks
Thank you
with regards
Harish Rotti
Ph.D Scholar
Department of biotechnology
Manipal life sciences centre
Manipal
________________________________
From: Igor Chernukhin <igorc from essex.ac.uk>
To: arrays from magpie.bio.indiana.edu
Sent: Monday, 6 February 2012 3:12 PM
Subject: [Arrays] microarray data analysis
Hi All -
I would greatly appreciate if you give me advice on microarray data
interpretation.
We have a custom tilling microarray from Nimblegen for a newly sequenced
genome of (some) organism. The genome is poorly annotated yet and all
probe annotations are in scaffold coordinates. It is dye-swap array
hybridized with mRNA from two different biological conditions. We have
processed the data with nimblegen commercial RMA software and also have
stats made with limma/vsn2 for each probe: fold difference (pmean), fdr.
The research group who studies this organism wants rather simple answer
like which transcripts are upregulated at which conditions and present
it in a ‘simple’ format. RMA output is not really helpful because it
gives all in scaffold coordinates. However we can see the difference in
probe values corresponding to transcript coordinates (annotated from
currently available gff) and of course it varies within the region from
significant to nonsignificant. Would it be appropriate to calculate the
mean of pmean values for each region corresponding to transcript (or
exons) and give it as the answer? Is there any other way of doing it…?
Many thanks
-Igor
_______________________________________________
Arrays mailing list
Arrays from net.bio.nethttp://www.bio.net/biomail/listinfo/arrays
