I think you must first confirm that your miRNA targets directly its putative mRNA target; hence you should use a luciferase reporter plasmid together with mimics/inhibitors. This is a very important step before proceeding to WB or real-time PCR. Actually, I would expect an effect on protein levels rather than the mRNA and we prefer to keep mimics into cells for a maximum of 24h. Shorter transfection duration reduces non-specific binding of miRNA mimics. Nevertheless, doing a time course for your cell transfection is necessary and try to optimize transfection conditions (mimic/inhibitor concentration, negative/positive controls, transfection duration etc) as such experiments are really sensitive and prone to give false positive results.

That is an interesting observation. But certain mirna actually stabalize mrna and allow more protein transcription, where as others block mrna and prevent transcription, or cause mrna degradation.

48Hours might be a bit too long, i would recommend using 5-6 hours transfection and then leaving it over night and collecting samples next day. Are you treating cells in any way? or is it just transfections?

Ok so first I think you should try a double transfection with your mimic. Plating, 1st transfection, 48h post transfection 2nd transfection . 48h upon 2nd transfection harvest and do a western. Make sure you are using the right antibody for your protein. Check literature. In addition if you see up regulation of mRNA of a target it may be cause an mRNA expression suppressor of the gene is being targeted by your microRNA therefor you see an indirect effect -increase on mRNA level. Let me know how it will turn out.

Some proteins like keratins are very stable and can last several days after knock-down. Check the stability of your proteins.

Dear Martina,

I did not understand. Did you see a decrease on mRNA level by mimic-miRNA overexpression and upregulation by inhibitor-miRNA transfection? If the downregulation of mRNA level after 48h is 10-20% is possible that you will not see any decrease on protein level due to the sensitivity of western blot. The qRT-PCR is able to read 10-20% of downregulation but by western blot often you need more than 40-50% decreasing and it is determined by the specificity of your antibody. If by qRT-PCR you read more than 30-40% of downregulation it could be a good idea to run your western blot after 24h, 48h and 72h of mimic post transfection. The turnover between RNA and Protein is not linear. For example if your mimic-miRNA works at 100% after 24h you will see a good downregulation on protein levels. On the other hand if the protein is too much and stable you need more time to see a downregulation by mRNA degradation. Personally, I am suggesting you to run the experiment at different time points: 24, 48 and 72h. Good Luck

I have observed something similar in my experimental approaches. I actually performed a time-course experiment with cells transfected with a miRNA mimic and I observed that the miRNA overexpression peaked at 24 hours and then the levels of miRNA decreased... I observed that the mRNA level of some putative target genes increased upon the transfection with the miRNA mimic. Surprisingly, they also increased with the respective miRNA inhibitors... I did not try western blot yet, because I'm trying to select the most confident target genes and I found that this is very difficult by analyzing the mRNA level. One possibility is to immunoprecipitate with anti-Ago2 antibody in your cells trasfected with the miRNA mimic and then to analyze the mRNA level of your putative target genes. You can expect that, if they actually are target genes, they would be enriched in the Ago2 IP fraction at the mRNA level.

SinceI have a similar problem... let me know when you resolve the question...

Excellent comments, another think to bear in mind, some miRNAs can up regulated rather than promote degradation of mRNA target. That's been observed when miRNA target the 5'UTRs. Check and see if your miRNA-mimics are not targeting the promoter region of your genes. Second, I always ask the people in my lab to do titration of transfected plasmids, miRNA-mimics. If the endogenous expression of mimic is hi in the cell that may cause compensatory downregulation of activity (either degradation or decreased processivity) for both endogenous and mimic miRNA and you'll se upregulated mRNA and protein, respectively. Third, I agree with Efi to check and see if you are not having off target effect, in that case you can easily observe a secondary as she mentions. Try to collect after 24 hours of post transfection or even 12 hours and check what you see. Finally, sometimes mimics are not very well incorporated in the RISC complex because they bypass the normal processing steps. If you can try also to use plasmids and see what's the effect. good luck:)

Sorry Lidia but you are speaking about putative targets....A miRNA can regulate 100-200 target genes and so you cannot exclude secondary effects...If you are not sure about your targets it is possible to observe these kind of perturbation...I know it because I worked to analyze and validate many putative target genes....You need to setup your experiment using known target genes...If you don't know it you need to test a list of putative target genes and see if they are down- or up-regulated after gain- and loss-of-function miRNA studies....

My thoughts exactly. Due to the ambiguity of the putative targets of miRNAs, it will be difficult to answer this question unless you are working with well known targets. It is interesting that you find that the mRNA level is upregulated by transfection with miRNA mimics, which does not lead to perturbation of protein expression. I am wondering whether it is the norm or exception.

I would be interested to know about the housekeeping gene that you are using for your qRT-PCR and if the expression of this gene is stable (i.e. within acceptable range of variation)? Sometimes the housekeeping genes might be regulated which might result into a false positive downmodulation of your gene of interest. Considering you have a genuine downmodulation of your gene of interest in qRT-PCR, your miRNA is targeting a secondary gene which is responsible for degradation of your gene of interest. As a result you have a highly stable protein in the absence of a functional degradation machinery.Let me know how it turns out.

I think you must first confirm that your miRNA targets directly its putative mRNA target; hence you should use a luciferase reporter plasmid together with mimics/inhibitors. This is a very important step before proceeding to WB or real-time PCR. Actually, I would expect an effect on protein levels rather than the mRNA and we prefer to keep mimics into cells for a maximum of 24h. Shorter transfection duration reduces non-specific binding of miRNA mimics. Nevertheless, doing a time course for your cell transfection is necessary and try to optimize transfection conditions (mimic/inhibitor concentration, negative/positive controls, transfection duration etc) as such experiments are really sensitive and prone to give false positive results.

hi guys

I myself have done three times luciferase assay on my target and it works really well I'm waiting to receive mutant UTR to be completely sure that it targets UTR.I have used time course studies of introducing miRNA(ambion) to N2Acells with different concentration of 50 70 and 100 after 24hrs and 48hrs.I saw once a bit downregulation but two times it was badly upregulated once after 24hrs once after 48hrs! I thought maybe here is a good place to discuss mine.Previously my colleague has shown that it works in primary neurons (target protein is downregulated there).I thought maybe conditions of experiment is important for example the media used during transfection the concenteration of serum or the hours we let cells exposed with miRs then we change media or really simple explanation.for example I know that my putative target is somehow involved in apoptosis not inducing it (not much published data) but I really dont have any idea how i should keep the experiment repeatable! plz help