Hello Everyone,
Currently, I am trying to silence a gene in a panel of CLL and MCL cell lines. In my first attempt KD efficiency ranged from 50 to 75% (assessed by qPCR, I have not looked at protein level yet) and there was no expected biological effect. I seeded 175k cells per well in 48well format and incubated them in Accell delivery medium for 72h, then I added FBS up to 10% and processed the samples after the next 24h.
My guess is that KD was not efficient enough to induce an appropriate response or target proteins weren't degraded yet. I'm going to repeat the experiment and incubate cells after the addition of FBS for 48h and then check the protein level, but I'm also wondering if a lower density of cells could increase KD efficiency? I used a lot of cells per sample to make sure that I'd get enough RNA, but I still can decrease the cell number.
But in general, I would be grateful for any tips regarding Accell siRNA as well as other efficient methods of delivering nucleic acids to lymphoma cell lines (excluding nucleofection - I tried it and this is a nightmare).
Thanks for the help!