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Here we discuss aelection and normalization of reference genes
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I was wondering whether anyone could help with this problem:
I want to compare the expression of a gene of interest between a mutant mouse line and wildtype controls. Only very limited quantities of RNA were available from these mice, as the lab no longer has access to them. Having quantified the available RNA, there was enough to measure three reference genes and the gene of interest. I chose three reference genes from different functional classess and with no a priori reasons for being regulated by the mutation.
I realise that three genes is really nowhere near enough, but I'm now wondering how best to use the data from these genes for normalisation. I have tried analysising my data using NormFinder and find that the genes have very poor inter- and intragroup stability values (the best combined pair have SD = 1.4).
Is there any point in normalising to these reference genes? And if so, would it be better to use all three, or to use NormFinder to identify the most stable gene(s)? Does it make the situation any better that the gene of interest shows a very large difference in Ct values between the groups with the direction of this difference being consistent across 6 biological replicates?
Any help would be much appreciated.
Seems you are in trouble. Either the selected reference genes are poor or the experimental protocol (incl. preanalytics) introduced large confounding variation.
Have you estimated the SD for the selected reference genes for the WT mice only by comparing with an established panel, for example: http://www.tataa.com/products/Gene-Expr ... ducts.html. If you get high SD for your assays in such comparison then probably something is wrong with the assays.
What is the accumulated SD calculated with Normfinder with the three genes?
Have you compared with total RNA normalization? The calculation is also based on Normfinder, see: Tutorial: Choosing a Normalization Strategy for RT-PCR GenEx System Aids in the Selection of Reference Genes for Standardizing mRNA Measurements. Genetic Engineering & Biotechnology News 28, 2008 (available at http://www.tataa.com).
2 posts • Page 1 of 1
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