We are using retroviral shRNAs (both tagged and untagged) and made glycerol stocks after getting them from Origin. Have used them a number of times with proper downregulation profile. But now they are not working or working sometimes as we are trying to downregulate. The tagged (RFP) one shows that transfection has worked but downregulation of the gene has failed. Is there any methodological problem done by us? Please suggest. Normally we are using Lipofectamine 3000 for the transfection.
Are you talking about stable cell lines or transient expression? If we are checking transient transfection changes, that can be varied by transfection efficiency, cell line type (mouse promoter or human promoter), plasmid quality etc. In case of stables the promoter inactivation by methylation problem with many passages might happen ( most of the down regulation vectors contain tag under additional promoter).
Sometime the sequence tend to loose. My recomendation is grow them in terrifci broth to isolate plasmid DNA, sequence theme to confirm the shRNA seq, and then carry out transfection. I also had similar problem before.
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