Hello,
I have a problem with the PCR results from my mesenchymel stem cells lysates. I use beta-actin as a reference gene and for some time now the Ct value is at the level of 30-35 cycle, a few months ago I did not have this problem, Ct was at the level of 15-16.
These results are passage 2, passage 6 and passage 12, everywhere a bad result which therefore does not depend on the passage.
All DNA concentrations were measured by nanodrop and reduced to one concentration, reverse transcription was performed under the same conditions. All probes were analysed on the same PCR plate at the same time. It is not the fault of the starters, because they were frozen in -20, used in the same concentration, on one plate. In new samples they showed the level of 30-35 (bad result), and in the old ones they are fine 15-16 (good result).
So what is the cause of such discrepancy in actin values?
What could this change result from? Is cell culture contamination possible resulting like this ?
"All DNA concentrations were measured by nanodrop and reduced to one concentration" -do you mean RNA?
Because you really shouldn't spec cDNA: this is largely meaningless (unincorporated nucleotides and primers all contribute to the absorbance).
If all else is equal, my suspicion is that cDNA synthesis was just...really bad for those specific samples. Have you looked at any other genes? If the cDNA synthesis was low efficiency, then everything will be low.
If this is the case, then my suspicion would be "dirty RNA", generally due to poor 260/230 ratio. Did you determine this ratio for all your RNAs?
Yes, I agree with John. Most of the time it will be the poor quality of the RNA or a problem with cDNA synthesis. Run a bleach gel electrophoresis (or alternatively with MOPS) to check the quality of the RNA.
Those all look fine. My cut off is usually 1.7, and you're safely above that in all cases.
Have you looked at any other genes in these samples? If they're low expression across the board, then I'd just chalk it up to "those cDNAs are terrible, for some reason", and make fresh cDNA samples.
If it's just beta actin that's weird, and only in those samples, then this could be a real effect. For what it's worth, I find that beta actin is a particularly labile transcript: in post-mortem samples beta actin is degraded much faster than most other mRNAs. Potentially if your lysates weren't handled identically you could see something similar.
I propose checking b-actin expression of different cell types and see the results. If they are the same then maybe your probes are degraded. Or maybe try to adjust the temperature.
I once faced the same problem when I used a different cDNA kit than the one that I used before and afterwards. I got it for testing purpose, was one that should write the cDNA pretty fast - but in the end, my samples were all crap.
To ensure the quality of RNA itself that it is not degraded, you can load it on an agarose gel.
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