# Virus titre and copy number of integration

If I need to preform a genome-wide high throughput screen using pooled shRNA library infection followed by deconvolution by deep sequencing, how would I determine the optimal infection rate at which the majority of cells only be infected with single copy of shRNA? Thanks!

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Do you mean infection, transduction, or transfection? If you mean infection, what virus are you using? Several viruses you can use an MOI of 1 to get that result, but others wouldn't produce it with an MOI of 10 (good example of the later is RSV). Presumably you are using a lenti system? – Atl LED Sep 11 '13 at 16:33
I also don't know what cells you're using. If your lab has never done lenti infections before, I recommend pLKO.1-puro optimization (or an analog) before trying shRNA library. That should give you a ball park MOI, but a good guess is between .5-3 – Atl LED Sep 11 '13 at 16:48
Yes I am using Lentivirus in human melanocytes. I need to get MOI of 1(one copy per cells) for majority cells for the purpose of later sequencing. I just need to determine the infection rate to achieve MOI of 1(is there asomewhat methematical model for this?) – Mai Sep 11 '13 at 16:49
Oh, are you wanting to know how to titer lentivirus to get the molecules or TU per mL? I didn't get that from your question. Calculating MOI is easy with the titer information. You have the number of particles/PFU/FFU/ELISA/RT-PCR readout per mL of virus stock. You then take the amount of virus you added to your cells (in what ever units) and divide by the total cell count. Perhaps I'm still not clear on what you want, but transduction optimization and titering are different things. – Atl LED Sep 11 '13 at 16:56
I did lentivirus infection before using pLKO system and I know the infection rate judging by GFP or puroR. However, we don't know the copy numbers of virus in each cell. The copy number of virus per cell is important when we are doing a pooled library screen-I don't want cells infected with copies of virus carry different genes. My questions is, by either mathematical or experimental approach, how we determine the optimal amount of virus added to cells so that the majority of cells will only carry one copy of virus while we still have a reasonable infection rate. Thanks! – Mai Sep 13 '13 at 10:55