Trouble with shRNA transfection
Posted 17 June 2013 - 03:26 AM
I need some help about shRNA.
Here is my story : I ordered shRNA plasmids from a commercial firm ( don't know if I can tell the name without being kicked of the topic...) to invalidate the receptor I'm studying. I made amplification and purification, then digestion to linearize the plasmid and transfected it in the cells.
After 24h-48h of rescue, I started a selection with puromycin at the minimal letal dose (I made a killing curve before, and obtained 0,1 µg/mL for one cell line and 1µg/mL for another). I had a massive death of the cell during one week, then growth of resistant clones.
I made a FACS analysis to compare the "target sh" versus Non" target sh" cells.................. and they have the same rate of expression, i.e no invalidation....The cells grow well in puromycin....
Does someone ever face the same problem?
I just see two possibilities : either I had no luck and selected "natural" resistant clones in the culture, which do not integrate the plasmid or the shRNA is not efficient (but I think the firm has validated the sh.....)
Posted 17 June 2013 - 06:00 AM
Edited by jerryshelly1, 17 June 2013 - 06:03 AM.
Posted 18 June 2013 - 01:58 PM
Thank you for your answers.