I have to perform expression profiling of microRNAs from serum samples.
As there is no universal normalization method yet available, I intend to use synthetic c.elgans miR-39 microRNA for normalization. This method involves generation of standard curve and absolute quantification. However, I am new to this method and I don't know how to perform absolute quantification and generate standard curve. Therefore, if there is anyone with personal experience or knows how to do these steps, can you share the appropriate protocol?
In May, we have published an article which aimed to evaluate the diagnostic value of a serum miRNA using both relative and absolute quantification. It's an article just about the method of quantification, so the protocol is explained in the text! If you can't download it from pubmed, I can send it by mail! For any questions, tell me!
Article Clinical significance of circulating miR-126 quantification ...
In May, we have published an article which aimed to evaluate the diagnostic value of a serum miRNA using both relative and absolute quantification. It's an article just about the method of quantification, so the protocol is explained in the text! If you can't download it from pubmed, I can send it by mail! For any questions, tell me!
Article Clinical significance of circulating miR-126 quantification ...
What I predict is As we use synthetic miRNA for normalization , the concentration will be known (which is based on nucleotide mass, by this indirectly we can estimate the copy number). At a X concentration probably x copy number will be present. By this way we can plot a standard curve.
Another paper that can help you in that question is:
Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10513-8. Epub 2008 Jul 28.
Circulating microRNAs as stable blood-based markers for cancer detection.
Mitchell PS, et al
In the supplementary matherial thay extensively describe both the absolute and the relative quantification method for circulating microRNAs and the generation of standard curves
Thank you everyone for your informative replies!!! . After reading mentioned papers I have understood the following. Please read and confirm if the steps are correct or not....For absolute quantification and standard curve generation, in addition to c.elegans miR-39 spike in, I also have to get synthetic versions of all those mature miRNAs which I am aiming to expression profile (for example miR-21, 221 etc)? At first, add c.elegans miR-39 at RNA isolation step in each serum sample, Then at cDNA synthesis step, prepare RT reaction mix for each serum sample, prepare serial dilutions of each synthetic mature miRNA and make their separate RT reaction mixes (as if treating each of synthetic miRNA as a separate sample) and run the cDNA synthesis cycle. Then at real time PCR step, take cDNA of each sample (disease, control, and from serial dilutions) and setup the PCR reaction... ???
Hi Azeem,
Have you considered doing relative quantification approach instead? Once you have your amplicons validated you don't need standard curves there - (it's still nice to have a few or at least 2 dilutions per sample, to check tor efficiency of amplification). In relative quantification approach you just select the "calibrator" sample, onto which you compare all the rest of the samples. You will still be able to see differences between all your samples but they will be relative (relative to your calibrator sample) instead of absolute copy numbers. That is still useful in most scientific questions. I did a screening with a larger number of samples and genes with qPCR in this way.
Is absolute quantification approach really necessary for your case?
Cheers,
Matjaz
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