Trying to express non-coding RNA in HeLa cells. Insert the 3UTR sequences into mammalian expression plasmids driven by CMV promoter. Plasmids should be no problem since both digestion pattern is as expected and sequencing shows no errors. However, qRT-PCR results didn't show increased expression of 3UTR using the RNA sample treated with DNase I, although qRT-PCR can show thousands times more expression with RNA samples not treated with DNase I. Any one can help me to figure out the problem? I doubt about the methylation of CMV promoter but curious about other possibilities.
There are few steps you might have to make sure: How efficient is your transfection? Did you clone the 3UTR downstream something? Do you have a positive control? I would suggest you always including a control i.e. everything else is the same but the part you cloned. Hope this would be helpful.
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