Hi all,
I am trying to perform a loss-of-function assay. I silenced some lncRNAs which I found them from a high-throughput screening considering changes in their downstream molecule. In order to validate, I performed a secondary screening in which I silenced those lncRNA candidates and I checked their downstream molecule via qrt-PCR and, it(the downstream molecule) is affected, I need to show how much I have silenced those lncRNA candidates. However, they seem they are not silenced, moreover some of them are upregulated upon siRNA treatment. Is there anyone who has a similar experience? Thanks
1-Primers are optimized. Checked melting curves and ran in an agarose gel, give a single band
2- Primers target a region which is close to 5' end or middle, not 3' end
Regards,
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