Validation of housekeeping gene expression - (Jan/12/2010 )

Hey there,

I would have a question on normalization of RT-PCR data. I want to measure the knockdown of TNF-alpha in murine RAW264.7 cells after induction with LPS and transfection with siRNA and different polymeric delivery systems.

I quite often have seen publications for the validation of the HKG expression in experiments using tissues so far. Do I have to do this validation for cell culture as well to determine the intergroup variation of several HKGs or is that not extremely necessary for the optimization?

Thanks for all answers in advance.

it is always required to validate any gene expression study by using a suitable housekeeping gene.
it is also ideal to validate the housekeeping gene itself, in your case, its HKG.

Hey thanks for the fast answer.

I think I phrased it a bit wrong. Of course I have to validate my housekeeping gene for expression stability.
What I was thinking of was if it makes sense to test out lets say 5-10 different HKGs and see which ones are the most appropriate. I am just asking if that makes sense also considering publishing this. Would the validation for one cell line be enough for a publication or do I need definitely some similar tissue samples or additional cell lines.

hi,

Currently there is a strong debate on how many house keeping genes should be used for normalization. To be safe, at least two to three house keeping should be used.

You should check the expression level of the house keeping gene (s) across all your control and treatment groups to validate that its expression is similar in all these groups.