My experiment is related with miRNAs. I would like to see the effect of miRNA on transduced cells. So the idea is that miRNA after causing obstruction of protein production should show subsequent effects on related pathways. My question is at what time point should I be able to see those subsequent effects overall? Has anybody done such experiment? I ll be glad to have an answer to this. Thanks
Hi Neeta,
I tested a few siRNAs for knockdown of a virus replication in a hepatic cell line. I checked the inhibition levels at 4h, 24h, 48h and 72h after transfection of infected cells.
At 4h time point I did not observed any change on my target expression, at 24h the target was inhibited at more than 70% (30% expression) and at 48h the maximum inhibition level was observed. I did not observe any difference from 48h to 72h. So, on my experiments, the maximum inhibition level was at 48h after transfection.
As miRNA and siRNA use the same pathway, I believe you should start observe the effects after 24-48h. But it really depends of which step of your pathway you will be analyzing the effects of your miRNA and the half-life of your proteins.
Hope I could have helped you.
My understanding from previous experiments and experiments conducted by others, is that optimal effects are seen on RNA levels 48 hours after transfection, and on protein levels 72 hours after transfection.
Thank you very much Bruno and Breanna for your answers..... hopefully I ll come back to give my feedback after I am done with the experiment.
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