Question: Normalization based on housekeeping genes
gravatar for elenichri
4.5 years ago by
elenichri10 wrote:

Dear Biostars team,

I have a gene expression dataset which is produced by Illumina HTv14 microarray platform. I have performed log-transformation and quantile normalization between samples based on all genes of the array. Now my  boss asked to see the absolute levels of a gene of interest, thus asked me to normalize based on a set of housekeeping genes. I know it is not the best strategy for normalization but i need to do that...and i have never done that before. I did some reading on it and found that i have to normalize based on the expressions of these genes. I am using R. Would a function like normalizeBetweenArrays {limma} or normalize.quantiles.use.targets {preprocessCore} be suitable,by setting as targets the expressions of the housekeeping genes? I tried it but i get totally different genes to be diff expr between the conditions than the ones I was getting using quantile normalization based on all genes of the array. Do you have any suggestions?

Thank you very  much!



normalization microarray R • 2.9k views
ADD COMMENTlink modified 4.5 years ago by andrew.j.skelton735.9k • written 4.5 years ago by elenichri10
gravatar for andrew.j.skelton73
4.5 years ago by
andrew.j.skelton735.9k wrote:

Normalising to housekeeper genes shouldn't really be done with HT12 arrays, as far as I'm aware. The recommended normalisation is VST, followed by RSN or Quantile.

Housekeeping genes are only noted here by Illumina in reference to Control Probes (which are mostly used for background correction).

A better suggestion might be to show the expression of the housekeeping genes, to reassure your boss. Here is an example of how to do that. These control probes should be used to gauge how successful the array was in hybridisation, staining, etc.

ADD COMMENTlink modified 5 months ago by RamRS27k • written 4.5 years ago by andrew.j.skelton735.9k

Thank you so much for these two resources Andrew. They are really informative!

ADD REPLYlink modified 5 months ago by RamRS27k • written 4.5 years ago by elenichri10
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