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 delta Ct value is at the level of 30-35 cycle, a few months ago I did not have this problem, delta Ct was at the level of 15-16.
I use the same methods, the same isolation procedure and reverse transcription assay of RNA, the same primer concentrations and so on.
What could this change result from? Is cell culture contamination possible resulting like this ?
Hi,
it seems you talk about Ct not delta-Ct values? Hard to say what causes this shift in Ct values. Were the shown data measured in the same qPCR run? Do you want to quantify gene expression or DNA copy numbers? If gene expression analysis: Have you measured the quality and quantity of your RNA and used the same amount in the different reverse transcriptions?
Best,
Norman
Hello
No, it is not culture contamination.
What is the passage number of the mesenchymal stem cells?
It is known that under extended cultivation there is a marked decrease in the content of proteins involved in cytoskeleton structure dynamics.
Increase in passage number leads to a senescent stage characterized by proteome alteration at the level of cytoskeleton organization, actin dynamics and decrease actin turnover.
This could be a possible reason. I suggest you use cells of low passage and re-check.
Best Wishes.
Hi,
We too faced similar issue. If you are sure about the quality of the probe and RNA, no need to change. Ensure, proper freeze thaw of master mix, probe and RNA. Keep a record of freeze thaw cycle number and document the intensity and Ct value of the target.. You will find a pattern. Like this, we did for a while and observed that the variation in Ct value (of internal controls, PC) is mainly related with Storage and freeze-thaw cycles only.
Hello!
As Malcolm already stated, the passage number plays an important role. As well as differentiation status of the mesenchymal stem cells. If you are doing differentiation studies, some reference genes change drastically after or during differentiation.
If you can, try different reference genes out and see which one works better for your experiment setup.
Best regards,
Astrid
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?
....I would also check the RNA quality (agarose gel, Agilent TapeStation etc.). If comparable between the old and new RNAs I would do a new RT with some of the old and new RNAs within the same experiment
Best,
Norman
Hello,
If you still have the old sample, please conduct the experiment again.Then, compare with the new one. You might be need to carry out the revers transcription RT for your new RNA again and make sure about the RNA amount that required for synthesis the cDNA. The RNA could degraded during the preparation.
Also, you need to prepare new primer working solution it might be contaminated and check how many time freezes-thaw the polymerase.
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