Question: Normalization according to reference genes
gravatar for cemilcansaylan
3.6 years ago by
cemilcansaylan0 wrote:


I am new to bioinformatics, I have microarray data that platform is [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array. I want to normalize expression for each gene relative to the expression of the five reference genes (ACTB [the gene encoding b-actin], GAPDH, GUS, RPLPO, and TFRC) in R. Is there any package that ı can use.

Thank you very much

ADD COMMENTlink modified 3.6 years ago by igor9.6k • written 3.6 years ago by cemilcansaylan0
gravatar for igor
3.6 years ago by
United States
igor9.6k wrote:

You generally should not normalize microarrays to housekeeping genes unless you have a very good reason to.

See full explanation here:

One early approach was to select one gene, or a small subset of genes, which were not expected to be differentially expressed across any samples; these were usually chosen to be ‘housekeeping ’genes: genes which are required for basic cell processes, and which were once believed to have very stable expression levels in all cells. This commonsense approach is used routinely in rt-PCR. However, these housekeeping genes seem to vary by 30% or more across healthy samples, and even more in cancer samples (Lee et al, 2002). A 30% error in normalization is not a big deal for rt-PCR, where the accuracy is at best a half-cycle, and one cycle corresponds to an increase of a factor of two. However, in a microarray study a 30% difference over the whole genome can give many false positives.

See previous discussion here: Normalization based on housekeeping genes

ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by igor9.6k

Thank you very much for your helping

ADD REPLYlink written 3.6 years ago by cemilcansaylan0
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