I have a gene X, it is an activator of different normal cellular pathways. If I use a control group, it should be highly expressive. And literature also defend this. I have use RT PCR, I use GAPDH as an internal control with Sybr green. My results are quite different, it has similar results for my control and disease group. In all groups the gene is down regulated. It is not possible for a regulatory gene to be down regulated in control group. If my internal control is fine, it shows good results, what I have done wrong with my procedure?
So you have two groups 1) Control, 2) Disease. In this you have two targets a)GAPDH and b) Gene X.
When you say your results are quite different can you expand on this? It sounds like when your GAPDH is fine then the results are fine and when the GAPDH isn't fine your gene of interest expression is low? Would that be right?
Are you using cell line or clinical material? Secondly is GAPDH the usual control gene previously cited and can Gene X effect it?
Would need some more details to really help you out for this but it is possible the regulatory genes can be down-regulated. If you can provide a bit more info that would be useful
Best
Stephen
Agree with Stephen
People think you can use GAPDH for an internal control for everything. Generally it does perform well but now with everytreatment or tissue
Whatever internal control the litersture uses you should use as well
Failing that you need to verify that housekeeper is stable with and without treatment
If you are going to assay GAPDH in this regard you msy as well try one or two alternatives that others have used successfully, e.g.
B actin
B tubulin
B2 microglobulin
As Stephen said, more details are needed to understand the actual problem you faced and forward possible solutions. And also how about the replication of the experiment? If it is just the first trial, please try to repeat the experiment with more care on the procedures as technical mistakes usually lead to wrong or opposite results. Good luck!
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