Hi there,
I received extracted whole RNA from TIPS patient serum and negative healthy controls. All samples contained spiked-in SV40 miRNA already as a reference gene. I performed miRNA reverse transcription and analyzed expression of my miRNA of interest during Real Time-PCR. However, it turned out that expression of SV40 differed tremendously between TIPS patients and healthy controls although it should be nearly the same in the end. Because I got this samples from a neighbour group for analysis I assume different handling of those samples e.g. prolonged freeze-thawing of one set of samples. As this situation complicates a comparision of miRNA expression between TIPS and healthy controls I wondered whether there is a reference miRNA in serum which I could use for normalization instead ? Did anyone quantify circulating miRNA from human serum before using an endogenous miRNA and not a spiked-in miRNA?
I am looking forward to your experienced responses.
Thanks for helping me out.
Hello Milad,
thank you for your answer. Would you be so kind to attach some references if possible. Is it then RNU6B that you used in your projects ?
Though mostly U6 is used as a reference gene in case of miRNA studies, but it has been observed that after a few freeze-thaw cycles the expression levels destabilize. Even then it is the most widely used reference gene. miR-16 also can be used as an internal control. Moreover you could also go for the old pal GAPDH.
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