If I measure gene expression in an organism under two different conditions, do I still need to normalize these genes to a housekeeping gene or comparison between the genes under two different conditions is sufficient?
I see the necessity of comparison of a set of genes to the expression of a housekeeping gene, but when the conditions are different and the amount of the cells re the same, if the expression is affected, why normalize different sets to respective housekeeping gene?
If you can ensure that
- the amount of cells is equal
- the efficiency of RNA isolation is equal
- the efficiency of the RT is equal
- the amount of cDNA subjected to the rtPCRs is equal
then you can omit any normalization.
However, it's usually rather difficult to ensure all these things. Its easier and safer to to rely on all that and simply normalize to a loading control.
Note that not every housekeeping gene is a valid reference gene (loading control). A reference gene must be expressed in the target cells and its expression must not be altered due to the treatments (that's not neccesarily true for housekeeping genes! Example: Gapdh is regulated by hypoxia and thus not a valid reference gene in experiments involving hypoxia; Actb is regulated upon cell migration, growth and division and is thus not a valid raference gene when such processes are involved).
Ideally use a whole panel on (putative) reference genes. You may so possibly identify genes that are regulated (which you may not use to normalize then) and/or individual (slight) changes in their expressions will likely average out, giving you more reliable results.
Thank you. I'll select the same gene as others did then. You are right, normalization sounds easier, although the crll quantities will be the same amount simply because they will be split into two equal parts.
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