No reduction in protein expr after 2nd passage of transfected cellsSilencing
Posted 19 September 2013 - 12:41 PM
Posted 19 September 2013 - 01:07 PM
shRNA can be tricky. The main thing is you do not know where the shRNA will intergrate into the genome. It is possible that the shRNA is integrated into inactive heterochromatin or recombination damaged the shRNA construct. This would maintain antibiotic resistance, but hinder shRNA expression. It is also possible that something is altering the formation of your shRNA. When you ordered the lentivirus, did they give you an information sheet with binding information? Sometimes this costs extra, but it would allow you to check possible binding sites of your shRNA.
Alot of things could be going wrong. It is hard to pinpoint just one.
Did you use a negative/positive control?
Did this help?
Posted 19 September 2013 - 07:54 PM
Posted 20 September 2013 - 06:38 AM
You should be able to. I have always had problems with shRNA expression when the passage becomes to high. I like using a GFP tagged contstruct to determine the effectiveness of the shRNA. Your method is fine though.