hello every one
I have transfected mda-231 cell lines by siRNA and lipofectamin 2000. siRNAS are against a long noncoding RNA, and have flourscent tags in their 5' end. 24 and 48 hours after transfection efficierncy of transfect by flourscent microscope is atleast 70 percent but real time PCR shows no change between normal and transfected cells. I have used 50nM 75nM siRNAs.by the way I donot design them by myself, they are commercial and bought from qiagen .
To add, how large are the molecules that you are targeting? It could be that you haven't waited long enough as larger molecules have longer half-lives. Also, if it is a commercial product, feel free to call their technical help department as they are usually very responsive.
For many mRNA targets, good knockdown is achieved at 12h. So 24-48h time points are good. siRNA concentration is very high, it should work; to avoid off-target effects, actually use lower concentration. Either siRNA is not potent, or delivery did not work (estimation by tracing dye is far from being perfect), or readout is suboptimal. Use a positive control- eg Silencer Select GAPDH siRNA, that under optimal conditions induces >95% knockdown as measured by qRT-PCR. If positive control works good- your in vitro delivery and qPCR readout are fine. Then its siRNA- try more designs
Nina Hahn
thanks for your answer, I am going to check after 72 hours and maybe in 12 hours too.
Paul Joshua Stirba
thanks for your answer Paul, the lncRNA has 1.5 kb length. it s expression is partly low , ct obtained from normal (untransfected) cells is 30.
Parichehr Maleki That seems somewhat large to me, I'm guessing it'll take longer than expected (by how much I am not sure).
Alexander Sasha Vlassov
Thanks for your comments, I think I should try 12 hours and 72 hours time points, we do not have siRna against gapdh, but there is allstars hs cell death control siRNA from qiagene availabe , it works 48-96 hours after transfection by observing cells by visible microscope according to manufacturer instructions. is it a reliable positive control? I have used that once before and it worked but I should check it again
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