I am study in transcriptional regulation.
Before we cloned gene promoter into reporter vector and transfected the reporter vector into rat intestinal cell plus another HIF expression vector to express Hypoxia inducible factor which upregulate this gene expression. Result shows 4-6 time induction compared with the cells without the HIF vector co-transfection.
Now, we want tot see whether the endogenous gene has similar level induction as the promoter luciferase result.
We transfected HIF expression vector to the cell and then isolated the total RNA, synthesize the first strand cDNA with bio-rad kit and run qRT-PRC with the primer used for a long time for this gene. Result shows no induction.
I do not know what is wrong with the qRT-PCR. Both methods used to measure the transcription level, how ever show different result.
Do you have experience like this? We doubted that we missed something however have not identified what we missed.
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