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 I used U6 as internal control in about 7 projects to identify more than 28 microRNAs and I suggest U6 . in some other projects we used snord genes as internal control .
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.
I dont know how I could send references here ,please write your mail , I will send you references
Hello Milad,
my email is [email protected]
I really appreciate your help.
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