That is an interesting topic. Technically, the inhibitor should not have any effect on the expression of the targeted microRNA. Often it does. That is very hard to understand unless the inhibitor somehow interferes with the PCR. So, technically you can't or should not use qRT-PCR to determine the activity of the inhibitor. It is still widely accepted as a measure of your inhibition. Would be nice to hear the opinion of other people.

If you want to be sure your inhibition works, you have to either check a few well established target genes (good luck with that!) or co-transfect a sensor for your microRNA (a perfect match sequence, so that the microRNA can work as a siRNA on this artificial 3'UTR). This sensor/luciferase assay should show 40-90% increase in luciferase activity after inhibition of your microRNA of interest. That sounds cumbersome but is one of the very few ways to be sure.

All this also depends on the level of endogenous expression of your favorite microRNA, transfection efficacy, and transfection toxicity. If your mciroRNA is highly expressed, you need mor einhibitor to get a decent repression of the microRNA activity. In that case 50nM may not be enough. If your microRNA is not in the top20 expressed you may be fine with 50nM or less. But these are just rough numbers. If you have a sensor construct, you can test this for yourself. The sensor will give you a rough idea in what range the majority of target genes may also be de-repressed (at least in a simple world).

Hope this helps.

If the function of your inhibitor is just to bind with miRNA rather than degradation, you may not see the difference in Ct value. Alternatively, you can check the expression of known target mRNA of that particular miRNA.

Thanks for your reply Mr. Ruhul Amin . Actually, I would like to suppress the endogenous miRNA using miRNA inhibitor and then to see the affect using the mRNA microarray. I am interested in finding the miRNA/mRNA targets. As I said before, I saw the affect with miRNA mimic.. but not with miRNA inhibitor. !!

You must also be weary of the toxicity implicit in the concentration of inhibitor you are using. Anything sufficiently high enough (in my case >50 nM) has the potential to be cytotoxic. You will have to optimize with varying concentrations if you have not already.

That is an interesting topic. Technically, the inhibitor should not have any effect on the expression of the targeted microRNA. Often it does. That is very hard to understand unless the inhibitor somehow interferes with the PCR. So, technically you can't or should not use qRT-PCR to determine the activity of the inhibitor. It is still widely accepted as a measure of your inhibition. Would be nice to hear the opinion of other people.

If you want to be sure your inhibition works, you have to either check a few well established target genes (good luck with that!) or co-transfect a sensor for your microRNA (a perfect match sequence, so that the microRNA can work as a siRNA on this artificial 3'UTR). This sensor/luciferase assay should show 40-90% increase in luciferase activity after inhibition of your microRNA of interest. That sounds cumbersome but is one of the very few ways to be sure.

All this also depends on the level of endogenous expression of your favorite microRNA, transfection efficacy, and transfection toxicity. If your mciroRNA is highly expressed, you need mor einhibitor to get a decent repression of the microRNA activity. In that case 50nM may not be enough. If your microRNA is not in the top20 expressed you may be fine with 50nM or less. But these are just rough numbers. If you have a sensor construct, you can test this for yourself. The sensor will give you a rough idea in what range the majority of target genes may also be de-repressed (at least in a simple world).

Hope this helps.

Hi Masood,

It depends on which kind of inhibitor you use. Some companies claim that the complex formed by the miRNA and the transfected inhibitor is stable up to very high temperatures. Therefore, if you use this kind of inhibitor and do not exceed this critical point during your reverse transcription, you will only work on "free" transcripts. In other words, you will not amplify the transcripts bound to the inhibitor during the qPCR step only if your complex is stable enough. So first check that the interaction between the miRNA and your inhibitor is not perturbed during your experiment.

You do not have this kind of problem when using a mimic as you artificially add the oligonucleotide of interest and do not work on complexes.

Something really important is that having a (non-)conclusive result by qPCR does not provide any information on the functionality of your transfection. Let's imagine that your complex is stable into the cells but is disrupted during your RNA extraction/RT/qPCR, you will not observe any change given the read-out you use but the inhibition actually happened into the cells and induced transcriptional/proteic response(s). In your case you would miss it. The opposite case would be a sequestration of your inhibitor into vesicles after transfection which would be released and allowed to bind to the miRNA after cell lysis. In that case, you would obtain a conclusive result by qPCR but your transfection would have not been functional.

It is very important that you look at the functional consequences of your transfection. You can follow Thomas Andl ideas for example. An other possibility would be to directly go for a genome-wide analysis and see if the transcripts you find deferentially expressed between your functional and control inhibitors/mimics are enriched in predicted targets. Maybe extreme but might worth the shot if you plan this kind of analysis later.