Firstly I would suggest you to have more endogenous miRNAs as controls( for example some suggestions are given here page no 9 in this pdf

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/100027897_TaqManAdv_miRNA_Assays_UG.pdf)

.There is no universal endogenous miRNA but any miRNA that has stable expression could be used as reference and you can report fold change of your target miRNA based on that miRNA (mean of many stable miRNAs) . Of course it is good idea to select the endogenous miRNA from the literature but it does not guarantee that it would be a endogenous miRNA in your experiment (But you can evaluate its potential).

Secondly people also use snRNAs such as RNU6B, RNU24, RNU44, RNU46, RNU48 as reference . I am not convinced with this approach . U6 also used but I wont vouch for it.

Using 3 to 4 endogenous miRNAs and seeing the same trend (Up/Down regulation) among all these endogenous miRNAs would give realistic findings

I agree with the suggestion to use multiple endogenous miRNAs as controls, and confirm the stability of expression. I would add that you can normalize to the geometric mean of multiple stable probes. The method commonly used is described in this manuscript: Article Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, D...

Working with serum, I might also suggest testing some miRs specific to RBCs or platelets just to reduce the likelihood of false-positives from the sample prep. There are a few publications that address this kind of contamination.

Ryan

Hi, Methee Sriprapun.

I do agree with others that you should have more than one endogenous miRNA for your study and, based on my experience, I would tell you to check the hsa-miR-16 (as you asked us) but keeping in mind it may not respond the same way in children as in adults or vice versa (you will need to check it by testing it in a few samples of your new study).

One question is:

From your study, If it was a mirNome or a large number of miRNAs, have you only found one miRNA that works as reference? If not, use all you found.

Did you use synthetic miRNA cel-miR-239b-5p as spike-in to account for possible inhibition of reverse tran-scription and/or PCR? If not, consider use it too.

Best wishes.

Hi!

It is important to use more than one reference gene and use the geometric mean of those to normalize your data. You can start checking the miRNA that resulted stable in the adult samples, but avery time you analize new samples you should tests your reference genes stability as it could change from one kind of samples to another.

Unlikely there are no consensus about stable genes across samples and diseases..so it is important to test a group of potential reference genes and select at least 2 using algorithms like normfinder.

Hi

We successfully used miR-103a, miR-20a, miR-21, miR-192 in human.