Hi guys, I found the mRNA level of oncogene （for example: C-MYC，C-FOS...) is unstable between different passgaes in hUC-MSC using realtime PCR. Is this a Q-PCR technical issue or is these genes are inherently unstable in hUC-MSC during cell passage?
Hello,
I would say it depends on several things.
First of all, how many passages have been in between your PCRs ? It is commonly known, that driver genes and/or mutations or in the beginning of the tumorgenesis needed genes may and can be lost during the development or as in your case cultivation.
Secondly it clearly depends on the genes itself, in my opinion.
And last but not least, it would be helpful to know how much difference you have in between your experiments as well as knowing how often you repeated the experiment.
Hope this helps you anyway.
Greetings,
André
Thank you for your response,André . My results are placed in the attachment, Cells are mesenchymal stem cells isolated from the umbilical cord and passaged in vitro. In theory, oncogenes are stably expressed, the expectation is that there is no significant difference in mRNA level between passages，however my results does not match, I repeated 3 times. Will it be related to the fact that the cells still not adapted to the culture conditions or Should I test more passages?
Hello Wang,
for me there are still things unclear to interpret your results.
First of all, what method did you use to generate your values? Did you use ddCT ?
Second, to which gene did you normalize your results or how did you normalize at all?
Nevertheless, for what I can see now, I would say you are looking at natural fluctuation because of the low values you are experiencing.
If you would like to verify your results you could probably look at genes known to be regulated by C-MYC or are regulating c-MYC (e.g. PVT1). If you have the time you could also take a look at the whole chr8q24 locus harboring c-MYC to see, if there is a genetic alteration or copy number variation leading to your result.
To answer your last question: No, personally I would not test more passages for the moment until you have clear evidence that it is not a natural fluctuation.
Hope this helps you a bit more.
Greetings,
André
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