As Thomas mentioned, you need to have a standard curve of your miRNA of interest. But as you say, this won't produce an absolute quantification if you still normalise to an endogenous control. Since you are using known concentrations of you miRNA of interest in the standard curve, you can easily calculate the absolute concentration of that miRNA your RNA sample.

However, the tricky bit is working out how to relate that to the absolute concentration of that miRNA in a living C. elegans. People often report the absolute concentration as copies per ng of total RNA. This is easy to calculate since you know how much RNA you used as input for the qRT-PCR. If you want to calculate copies per cell, then you need to make an assumption about the average total RNA content of a cell, and calculate from the copies per ng total RNA accordingly.

There's a detailed discussion of this and other issues with absolute quantification at: http://www.gene-quantification.com/absolute.html

One other point that may be of interest. When I was making miRNA standard curves for qRT-PCR a few years ago, I found serial dilutions of DNA oligos produced results indistinguishable from RNA oligos. I'd always assumed that the reverse transcriptase would have a higher efficiency for RNA, but for RT of miRNA mimics using stem-loop primers this wasn't the case in our hands. This saved a lot of money when assaying several miRNAs. I think we put it in the supplementary data of our paper if you're interested. 

http://www.gene-quantification.com/absolute.html

Article miR-29a and miR-29b Contribute to Pancreatic -Cell-Specific ...

I have used serial dilutions of both synthesized miRNA mimics (duplex) from our experiments and the C. elegant control miR for in vivo and in vitro AQ.  

thanks so much Thomas! but, if I put a serial dilutions of mimics and C.elegans, how can I know absolute quantities of miRNAs i am interested? C.elegans indicate the number of copies you have per volumen? Exist some formulation to calculate it? I'm so sorry but is the first time I have to do it and I don't know how calculate the absolute quantities not the relative infront of a housekeeping

As Thomas mentioned, you need to have a standard curve of your miRNA of interest. But as you say, this won't produce an absolute quantification if you still normalise to an endogenous control. Since you are using known concentrations of you miRNA of interest in the standard curve, you can easily calculate the absolute concentration of that miRNA your RNA sample.

However, the tricky bit is working out how to relate that to the absolute concentration of that miRNA in a living C. elegans. People often report the absolute concentration as copies per ng of total RNA. This is easy to calculate since you know how much RNA you used as input for the qRT-PCR. If you want to calculate copies per cell, then you need to make an assumption about the average total RNA content of a cell, and calculate from the copies per ng total RNA accordingly.

There's a detailed discussion of this and other issues with absolute quantification at: http://www.gene-quantification.com/absolute.html

One other point that may be of interest. When I was making miRNA standard curves for qRT-PCR a few years ago, I found serial dilutions of DNA oligos produced results indistinguishable from RNA oligos. I'd always assumed that the reverse transcriptase would have a higher efficiency for RNA, but for RT of miRNA mimics using stem-loop primers this wasn't the case in our hands. This saved a lot of money when assaying several miRNAs. I think we put it in the supplementary data of our paper if you're interested. 

http://www.gene-quantification.com/absolute.html

Article miR-29a and miR-29b Contribute to Pancreatic -Cell-Specific ...

omg!! Super-complete response haha thanks so much!! it has taken a long time for you!!! ^^thanks!

I agree with Timothy.

You may use the cel (or other suitable control mir) as an endogenous control target.  Spike the cel mir into a suitable volume of lysis buffer (trizol, et al) before lysing the cells. 

Normalizing to a spiked target could be expressed as:

I think this formula could be used: ((Copy #target)/(copy# spike))/(input RNA mass or cell number).

When validating the assays, a standard curve of the miR spiked into total RNA (6 to 8 log2 dilutions: 2ug, 1ug, 500ng, 250ng, 125ng, 62.5ng, 31.25ng...) start from untransfected cells should better reflect the RT-PCR assay conditions.

From the standard curve, determine copy number. You may express the result as (Copy #/input RNA mass) or (copy#/input cell #).  Untransfected and mock transfected controls should be included during validation and the experiment.

Consider the miR/mimic synthesis source and proprietary modifications, if any (Google patents should provide helpful information). Different vendors conjugate different chemistries to their miRs, which may affect RT, PCR or recovery efficiencies.  (I believe Dharmacon uses cholesterol).  

Really, thanks so much!!!!!! 

Dear Timothy please share supplemental material of your publication miR-29a and miR-29b Contribute to Pancreatic β-Cell-Specific Silencing of Monocarboxylate Transporter 1 (Mct1).

I could not get it from the provided link and from journal site.

[email protected] this is my email id

I recently published in detail a method to perform absolute quantitation of miRNAs. Might be useful to people looking to apply this technique and not sure where to start

Thank you so much