I harvested some lentivirus from 293T cells and want to titre the result. I infected 293T cells on a well plate with 400,000 cells per well which I infected with virus stock, and 1 in 10, 100 and 1000 dilutions (as well as a few uninfected wells). After 72h incubation I trypsinised the cells and used FACS to titre first, but 293T do not express the promoter under which GFP is located in some samples and hence these do not appear on FACS. As an alternative, I extracted the genomic DNA from all samples using a QIAGEN DNeasy kit (also the samples which I was able to titre by FACS already) and then performed qPCR on them.
I used the ABI PRISM 7000 SDS: http://www.cgenetool.com/products/abi_prism_7000.shtml
Included on the PCR plate were:
- Number standards: 1000, 10k, 100k, 1m copies
- Samples in triplicate
- No-template controls as well as untransduced controls
Each of those was doubled, once with primers and probe for WPRE (an element specific for the lentivirus) and once with primers and probe for beta-actin (a housekeeping gene present in all cells).
The results returned by the machine include for each well the cycle at which the threshold fluorescence was crossed - along with the automatically calculated quantity of copies (which the software calculates from the standards I assume).
Since I named the triplicate samples identically, it also automatically calculated the mean quantitiy among each triplicate (practical, eh?). I checked if there were any outliers which should have been excluded from the means but everything was fine.
So now for example I have a result of 700k for the quantity of WPRE in sample A. The quantity of beta-actin in sample A was 6.82 x 106. The transfection was 400,000 cells per well, sample A was transfected with 10uL of viral stock diluted 1:100.
How do I calculate the virus titre per mL from this?
I have been given the formula: WPRE Qty / (b-act Qty*0.5) * cells in well (400k) * dilution factor (100), but am struggling to figure out a) why b-act Qty is divided by 2, b) whether the result would be virus titre per uL or per 10uL (transfection volume) and c) how beta-actin can have almost 107 copy numbers consistently across all samples when there were only supposed to be about 105 cells...