We used Inducible TRIPZ™ Lentiviral shRNA (TETon) for transfection of HEK293T cell line. Although transfection efficiency is about %85, even GAPDH as positive control was not knockdowned. Is there any idea about this problem?
Hello Ozge,
is tRFP expressed after induction with doxy ? are 293T cells stably transfected and resistant to puromycin?
yes, the transfection efficiency was about %80-85 but we didn't puromycin selection for 293T
Probably I'm missing something..if you did transfection (i.e. no lentiviral transduction) and you didn't select for puro resistance you ended up in a transient transfection..is that so?
firstly thank you for your interest Dear Rocco Piazza,
but we take these photos and then directly lyse cells for protein extraction after 24 hours from doxy treatment. but is still transient transfection possible ?
Hello Ozge,
as a first test I would:
- increase the time between doxy treatment and cell lysis: detecting silencing at protein level is also dependent on target protein half-life. For instance, as far as I know, GAPDH half-life is approx. 35 hours. Therefore to start detecting a significant downmodulation you should reasonably wait at least 48 hours from doxy induction.
- try to assess target mRNA level by mean of Q-PCR before/together with protein analysis. Effect on mRNA is faster.
While doing these experiments I would also move to a stable line by puro selection.
thank you again your valuable advices, if i begin the experiment from first step, i will take into account this knowledge. but i have no chance for performing qPCR for this experiment
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