Hello miRNA experts,
I am doing a miRNA Taqman assay. The Ct values for all miRNA candidates are fine. But I have a problem with reference miRNA (Control ), I used three different controls: RU6B, RNU44, and RNU48. All of them are not stable; they are up or down-regulated. I also did serial dilution for the cDNA. The result still the same. Any suggestions?
What do you suggest the problem could be? I am not too fond of using only non-microRNAs as normalizers since they are "much" bigger than microRNAs. Therefore, they are differentially affected by degradation. It almost seems that microRNAs are completely resistant to the general RNA degradation process. If you look at a degraded samples and a samples with RIN value 10, the "mircroRNA peak" is basically intact and unimpressed by the destruction of non-microRNA RNA.
Therefore, one reason could be, that the quality of your RNA samples is not the same and some have more degradation than others. Then the question is: what do you mean with "All of them are not stable they are up or down regulated." 3-fold, 1.2-fold? If your expectation is that stable means they are in a 10-20% range, you may expect too much stability. Do all three of them go in the same direction, all three being simultaneously, coordinately up or down or are some up some down in one sample?
The next question is, do you analyze cultured cells or tissue material. I personally think the small non-microRNAs are good for cell culture with samples of good and consistent quality. From tissue material, I prefer to have some microRNA normalizers as well.
Finally, your normalizers may not always fit the idea of a normalizer. Perhaps some of them are not ideal in all contexts? If you use the mean of the three normalizers, how does that work out?
More questions than answers... But troubleshooting can be tough with limited information...
What do you suggest the problem could be? I am not too fond of using only non-microRNAs as normalizers since they are "much" bigger than microRNAs. Therefore, they are differentially affected by degradation. It almost seems that microRNAs are completely resistant to the general RNA degradation process. If you look at a degraded samples and a samples with RIN value 10, the "mircroRNA peak" is basically intact and unimpressed by the destruction of non-microRNA RNA.
Therefore, one reason could be, that the quality of your RNA samples is not the same and some have more degradation than others. Then the question is: what do you mean with "All of them are not stable they are up or down regulated." 3-fold, 1.2-fold? If your expectation is that stable means they are in a 10-20% range, you may expect too much stability. Do all three of them go in the same direction, all three being simultaneously, coordinately up or down or are some up some down in one sample?
The next question is, do you analyze cultured cells or tissue material. I personally think the small non-microRNAs are good for cell culture with samples of good and consistent quality. From tissue material, I prefer to have some microRNA normalizers as well.
Finally, your normalizers may not always fit the idea of a normalizer. Perhaps some of them are not ideal in all contexts? If you use the mean of the three normalizers, how does that work out?
More questions than answers... But troubleshooting can be tough with limited information...
Thank you Dear Thomas Andi and Nadia,
I used Pancreatic Cancer cell line treated with drugX and I selected some important miRNA candidates from microarray data. I also tested the quality of the RNA which it was good.
The problem in qRT-PCR.. the Ct values for miRNA candidates is ok and I can get they are up or down regulated in this Cell line but the normalizer are not stable.
for more detalis I treat the cell line with Drug X at different time points 0, 30 mins , 24 h and 96h. the 96h it is the most critical points for the treatment.
The most surprisingly, it is the normalizer regulated at 96 h point.
Where is the problem ?
I used the same RNA conc. like the other time points
the same cDNA conc. all the condition are the same !
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