I have been transducing MB-231 cells with lentiviruses carrying a GFP-miRNA construct. The cells showed good GFP fluorescence but RT-PCR does not show any overexpression of the miRNA. RT-PCR has worked for other applications. Any suggestions for a workaround or ideas as to what the problem might be (assuming there's nothing wrong with our RT-PCR protocol)? The lentiviruses were constructed using the Invitrinogen Block-it HiPerform Lentiviral Pol II miR RNAi system.
Having GFP expression do not means miRNA expression as they come from different promoters. When transducing cells, I always chech for the miRNA mature form, because if you check for the pre/pri-miR you will never know if the miRNA has been correctly procesed in the cell type you are ussing. Depending on the cell type and pre-miRNA promoter the immature miRNAs will be correctly processed or not. Forget about transduction efficiency (GFP). If you do not detect the mature miRNA in the cell, you will never see a phenotype. So I bet that probably those cells are not correctly processing your immature miRNA sequence.
Is your PCR recognizing pri, pre or matured miRNA? The miR RNAi system usually contain the precursor form. If your design is targeting primary transccript, you may not be able to detect it.
Having GFP expression do not means miRNA expression as they come from different promoters. When transducing cells, I always chech for the miRNA mature form, because if you check for the pre/pri-miR you will never know if the miRNA has been correctly procesed in the cell type you are ussing. Depending on the cell type and pre-miRNA promoter the immature miRNAs will be correctly processed or not. Forget about transduction efficiency (GFP). If you do not detect the mature miRNA in the cell, you will never see a phenotype. So I bet that probably those cells are not correctly processing your immature miRNA sequence.
Do you have high level of endogenous expression of that miR? Cell processing capacity may be an issue as well. We had such experience before.
You may start designing stem-loop primers in order to detect mature miRNAs using a real time PCR system, and also sequence your construct.
Which promoter do you have for the miRNA expression, CMV or EF1a? What does MB-231 mean cell type wise? Who designed the exact miRNA construct, Invitrogen or you? If I see the vector map correctly you have the miRNA expression cassette in the 3' UTR of the EGFP?
Potential problem: EGFP has a long halflife and will accumulate even if the promoter is only active very little. CMV is silenced in a lot of cells when the cells are not stimulate to have high NFkB levels. And the construct is having a competing mechanism between EGFP export/translation (needs polyA) and miRNA processing (cuts off polyA). So having a high EGFP expression can mean your miRNA is not processed at all. In which case it could help to know who designed the exact miRNA expression unit and see if you can tinker there.
Ps: I see only now that you replied that your RT-PCR targets the primary transcript and not the mature miRNA. That is not a good idea. Look for Applied Biosystems miRNA assays with TaqMan, as Ali above me stated, they work with stem-loop primers and detect the mature miRNA. That is very sensitive and should help.
A mature miRNA would mean he has to use a precise transcribing promoter and is loosing the incoporation bias from the pre-miRNA. Thus he would end up with a shRNA problem in terms of processing and loading. So that would not be helpful in my opinion.
Thank you so much for your input, everyone.
I have mentioned wrongly about my PCR. My PCR was actually testing for mature miRNA, using stem-loop primers, Taqman etc like what Ms Jung and a few others have mentioned. I am now testing for miRNA overexpression at different time points using the same lentiviral kit I've been using, since the expressed miRNA might not last long (as what has been mentioned also).
At the same time, I am also testing out another miRNA kit which requires transfection (an expression construct, not lentiviral) as my backup, in case my lentiviral kit is really not working for my breast cancer cell lines.
Check the genomic DNA for the miRNA sequences. Sometimes, the recombinant sequence contains a fortuitous regulatory element (ie splicing signal, other), which would limit its expression after the vector undergoes RT/integration.
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