Hello,
I am working on microRNA profile on human serum and tissue and need to normalize specific microRNA to a HKG in real-time-PCR, and for what I been reading miR-16 and Human U6 (RNU6B) are recommended.
I also been trying to find the sequences to prepare the oligos but I was not able to find them. Anybody has a recommendation to use as HKG in serum and tissue that works for real-time-pcr, and the sequences?
Please let me know. Thank you.
Hi Carlos,
Actually, most studies suggest that RNU6B is unsuitable for normalisation in many different cell/tissue types, including serum (e.g. 10.1093/bib/bbv056). As you mentioned, miR-16 is abundant in the circulation and has been used for exosomal miRNA quantification (10.1371/journal.pone.0183435). In this study the authors used multiple normalisation determination software to determine the best candidate. However, it is worth noting that they only examined a small panel of candidates based on prior studies. If possible, normalising to multiple miRNAs would probably increase the robustness of your data. Another alternative would be to use an (synthetic) miRNA spike-in control.
Josh
Joshua Clayton
Can you please tell me, if I used miR-39 as a spike in control beside miR-16 , How can I calculate the RQ ?! Shall I take the average of both exogenous and endogenous controls or what !
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