Hello,
i am a bit curious about my results. I transfected the human cell Line ARPE-19 with a Plasmid containing a GFP tagged p53 (p53-GFP, Plasmid #11770). After 24h i checked under the flourescence microscope and saw that the Plasmid was expressed due to the flourescence i could observe. (I also did a FACS previously to check my transfection rate which is about 50%).
However after i collected the cells, and did a PCR to check it there, there was no increase in p53 mRNA compared to an untreated control and to a sample i transfected the same way with the same backbone also expressing GFP but no p53 (pEGFP-N1-FLAG, Plasmid #60360).
Does anybody have an idea why there is no increase in the PCR compared to the controls ?
Thank you very much for your help, i really appreciate it.
Best,
Peter
Can you provide a some more details on your RT-PCR :
- Is this a SYBR RT-PCR or Taqman assay?
- Did you simultaneously check the expression of GFP?
- Was the expression of your house keeping genes around typical values?
Dear John Hardy Lockhart ,
it was a SYBR RT-PCR and yes i checked the GFP expression in all samples!
The cDNA was totally fine. So was the housekeeping Gene !
Thank you for your help
Peter Wolfrum
The first thing that comes to mind would be that your p53 primers are incorrect. However, that would require one of your primers to be specific to one of the truncated splice variants of p53. I'd double check the alignment of the primers against just to be sure.
You could try running the p53 wells out on an agarose gel to double check that the product from the RT-PCR is the expected size.
You might also try running a regular PCR on the plasmid itself using your p53 RT-PCR primers to verify that they recognize the plasmid.
As an alternative, try checking the change in p53 expression by Western blot. Because it's a fusion protein, you should be able to see the exogenous p53 running slower than the endogenous. You should see the same effect with GFP when comparing the p53-GFP cells to the GFP cells. If you can do double labeling on your Western (using fluorescently-labeled antibodies), you can confirm that the band is identified by both a p53 antibody and a GFP antibody.
thank you very much sir!
I will do a Western Blot probably next to see it on protein level!
How high is the transfection efficiency? Is it diluted by untransfected cells?
Transfection efficiancy is around 50 %, so the effect definitely should be seen!
Thanks for your answer :)
You can also do real-time of GFP as a control, and maybe if you have primers for any p53 regulated downstream genes like p21, that can also be examined.
Good Luck.
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