I used shRNA plasmid for the knockdown of gene. Firstly, package the virus, then transfect the target cells and then puromycin selection to get the stable cells. After selection of stable cells i did qPCR to check the knockdown efficiency and the gene was successfully knockdown. But the problem is that when i passage on the stable cells to get more stable cells, and then again checked the knockdown efficiency before sending the RNA for transcriptome sequencing i didn't got the same knockdown. I don't understand that what just happened in 2,3 days that i didn't got the same knockdown? Your kind suggestion will be highly appreciated.
Thank you
Two possibilities based on my experience:
1. Your stable cell lines have some non-transfected resistant cells growing slowly with your selected cells in antibiotic media.
2. Have you kept your cells under antibiotic selection media while passaging? Removing antibiotics from media can also allow non-transfected cells to grow and hence reducing your knockdown efficiency.
I would suggest the clonal selection of your cell line or performing FACS if the plasmid is expressing some tag. This would allow you to select only those cells which are expressing your plasmid gene or showing stable knockdown.
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