Determining transfection efficiency - (Dec/22/2005 )

Hi folks!

Please tell me some protocols to determine the transfection efficiency for a particular cell line and also sources of such protocol(s).

Waiting eagerly

What I do to dermine my efficiency of transfection is just using a plasmid containing a reporter gene (gfp for instance), perform transfection and the day after see how many cells have reporter gene expression.

For instance, when transfecting Hela cells, on day one I plate different amounts of cells, the next day I transfect using different amounts of DNA and transfection reagent, and the day after I check gfp with fluorescence microscope and if I see positive cells I wash my cells twice with PBS, trypsinize, wash again with PBS, and fix my cells (for all conditions I use the same volume to fix). Then I check how many cells had expression using FACS. The ones that have the highest ratio of gfp-expressing vs non-gfp-expressing cells are the most efficient, bearing in mind of course the survival rate (which I determine by the counts/second during the FACS experiment as I use the same volume to fix my cells, I take for granted the fact that dead cells aren't counted due to extensive washing before trypsinisation, which probably isn't 100% precise, but this way is close enough for me). To be more precise, you should also do a "real" viability count...

If you don't need exact transfection efficiency but just estimation, the simplest thing is to transfect a reporter (GFP, LacZ or other color reporters) into you cells, do a DAPI staining, and look under fluorescent microscope to get a rough count per field - efficiency is GFP cell# over DAPI cell# x100%. You may need to count more than 5 fields to get more accurate number.