Hi Ahmed Al-Omar

If you would like to take a decision about the housekeeping protein you have to take the following assay:

Let us suppose that you want to test the effect of X agent on the expression of a gene of interest.

Here, you have to treat you cells with X agent and collect RNAs at different times (ex: 0 time, 6 hours, 12 hours).

Then normalize the results of housekeeping proteins to the its 0 time and plot your chart. If you noticed high difference in the resulted results then you know that the housekeeping protein is affected by the X agent and it can not be used as a reference.

For more details please refer to:

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8. doi: 10.1006/meth.2001.1262. PMID: 11846609.

The best housekeeping is the one with the smallest variations between samples. Depending on the condition this can change the housekeeping expression, I believe you should choose the one with the least variation

Hi,

it might be better practice to test a set of potential HKGs, assess their stability via geNorm, Bestkeeper or similar and use at least two HKGs for normalization.

Best

Hello Ahmed Al-Omar

Appropriate validation of internal references is crucial to avoid misinterpretation of study findings. Validation of housekeeping genes are highly specific for a particular experimental model, and forma a crucial part in assessing any new model.

You will have to make a proper choice of internal reference that has minimal variability. You could include multiple reference genes which could help stabilize gene expression level calculations from RT-qPCR data.

For more information on how you could improve reproducibility and robustness of RT-qPCR-based gene expression analyses, please refer to the article attached below.

Article An Extended ΔCT-Method Facilitating Normalisation with Multi...

Best.

Thank you all for your helpful answers. :)