Hello, I'm struggling with my qPCR results, as I'm trying to show decreased production of microRNA in microRNA knockout cells (HEK cells). Which reference gene should I use in the qPCR experiment if these cells cannot biosynthesis microRNA?
Thanks ahead
I read in this article that this is the worst reference gene to use: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259703/pdf/srep41127.pdf
You have to validate the reference gene just as they have done in the article you posted above. I've also found U6 to be unstable in my samples (ventricular) during my doctorate studies. If you are using HEK cells, why not check some studies that have employed NGS with HEK cells and see the microRNAs that they detected. Perhaps this database will serve as a useful starting guide for you:
http://guanlab.ccmb.med.umich.edu/mirmine/help.html
I would recommending surveying about at least 5-6 genes and conducting a reference gene stability test as per the article you cited.
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