Does anyone happen to know any recommendation or experience for internal control microRNA (miR) for qPCR?
I tried to quantify certain set of miR extracted from human plasma samples.
Based on the previous literature and array-based expression data of our own samples, I chose hsa-miR638 as an internal control, but it does not work well mainly because there was a significant difference in miR638 expression level between our experimental groups, which is not regitamate (acceptable) as control, I think.
So I need to pick other internal control.
I also tried external spike in control, but because of the pretty low amount of input, it also did not work well. There was huge variance of externally input RNA.
Any comment or your experience are very appreciated.
thanks,
Masaki ITO
Wooo...
Honestly speaking, I have not perform circulation miRNA experiment you want to know. My colleague in Baylor performed CSF microRNA experiment to distinguish between germinoma and non-germinomatous germ-cell tumor, but he always said "There are no useful internal control!". I just remember.
Anyway, I cannot provide any information about your question. Very sorry...
Hope you have great luck and successfully make it your work :)
SY
Hi
we performed miRNA expression study from Sera of patients with Multiple sclerosis and healthy controls. We used cel-mir-39 as a spike-in control and it worked fine (helped to control for technical variation and also to evaluate the efficiency of extraction / reverce transcription / PCR). Also we used endogenous controls SNORD68 and RNU6-2, for this project they worked fine, but overall finding a good endogenous control for serum/plasma studies is quite challenging.
During the study we found that miR-21 was quite stable, however this miRNA is found to be deregulated for example in heart disease and cancers, so not sure if it is a suitable as control.
BR
Julia
P.S. personally, i found this article helpful, regarding the normalization techniques:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267743/
Hello Masaki
I advise you try with miRNAs as miR-223, miR-146a or miR-126. Because, its transport is related with lipoprotein particles. They are taken as internal control too. You should test a group of them and according to sample choice the more unvariable. It is a option whether your spike control does not convincent you.
Check this file
Best regards
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