Like Francisco J. Enguita said,  if you are talking about whole blood, then probably RNU6B would be suitable.  However, if you are talking about circulating, free miRNA in serum, or plasma, that would be a different story.  Although many miRNA can be detected, I don't think a clear consensus exists for which miRNA, if any, is suitable for use as a reference.  Spike-ins are probably the most appropriate at this point.

I have always used RNU6B as a control for miRNA expression by qRT-PCR. The life technologies website has good information on quantifying miRNA. Some of the other genes that you listed are also recommended. 

http://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/microrna-studies/tech-notes/finding-the-best-endogenous-controls-for-real-time-quantitation-.html

Hi Behnaz

I suppose that you are talking about blood and not about plasma or serum. If you are dealing with whole blood you can try some RNUs our tRNAs, or even to spike in your samples with a synthetic miRNA before extraction.

Best. Paco

Like Francisco J. Enguita said,  if you are talking about whole blood, then probably RNU6B would be suitable.  However, if you are talking about circulating, free miRNA in serum, or plasma, that would be a different story.  Although many miRNA can be detected, I don't think a clear consensus exists for which miRNA, if any, is suitable for use as a reference.  Spike-ins are probably the most appropriate at this point.

Thanks Nina . Are you working with human blood samples ? or cell line

Hi Francisco J., I already isolated total RNA ( containing microRNA) from whole blood.

Is there any difference between miRNAs isolated from plasma, serum and whole blood ? 

Many thanks

Hi Jon, Could you please tell me what is the difference between miRNA in plasma , serum with whole blood ? 

You mean, the total RNA (include miRNAs ) that I isolated from whole blood are different from those that are in plasma, serum ?

Thanks

Hi Behnaz,

I have used RNU6B for both cell lines and PBMCs isolated from whole blood.

When you isolate RNA from serum/plasma (as opposed to whole blood), you are collecting cell-free RNA which is made up mostly of very very small RNAs

Hi Benhaz, since circulating microRNAs are the results of apoptotic cell released, RNU6 is not a good idea for plasma and circulating mir. It is a good endogenous for cell-derived microRNAs. If you have to analyze mir from plasma, than i can suggest you the more stable mir-103a-3p and 93a. Anyway, try to use always at least a couple of endogenous instead of one, and if you can, buy the primers of 3-4 endogenous (if you want to try the RNUs, ) and with normifinder/genorm look at the more stable in your case study. Hope it helps.

Good luck

Claudia 

Hi Behnaz,

Whole blood consists of red cells, leukocytes, platelets and plasma.  So RNA isolated from whole blood will contain RNA from each of those components.  RNA isolated from plasma (anticoagulated, separated whole blood) is cell-free RNA that can come from dead/dying (apoptotic, necrotic, and trauma injured) cells, or be secreted from living cells in vesicles (exosomes).  As said by Claudia and Nina, these are usually small RNA, including miRNA.  RNA isolated from serum is a bit different, as serum represents the fluid phase separated from coagulated blood, which may contain RNA from activated platelets (platelet releasates) and cells lysed during coagulation.

RNA isolated form any source needs to be quality controlled to make normalization even possible.  Qiagen has good resources on how normalization can be carried out in these various sample types.

I hope this helps!

 Hi Claudia, Thanks for your suggestion. 

Hi Jon,

Thanks for your detailed explanation .

This is a constant concern. I would definitely use at least 3 controls (the ones mentioned above that show very similar levels in your different samples) then use the Ct geometric mean for normalization. And adding a spike-in is always a good idea to control for RNA extraction and  RT efficiency.