Calculating siRNA Transfection Efficiency - (Jan/22/2012 )

Hi all,

I am going to Transfect siRNA in primary Chicken embryo fibroblast. Please suggest me how to calculate the transfection efficiency.
I ve read about using fluorescently labelled siRNAs like Alexa flour. But presently our Flow cytometer is not working. please suggest me an alternative as i am nearing my year end of my research and i have to finish my work.

THanks....
VetVinoth

You can also view the transfected cells under a microscope to determine how many cells are fluorescence positive.

Thank you for your reply,

i also thought of counting cells but regarding calculating transfection efficiency please suggest weather i can calculate by this formula


(NO of Cells showing fluorescence in a field / total no of cells in a field ) * 100

or any other method is there...


Vinoth

That formula will give you a percentage, so it is correct for that method. If you wanted to do a lot of work determining the baselines and fluorescence levels for particular percentage transfections, you could use a fluorescent plate reader to determine the efficiency.