Dear All,
I'm having trouble figuring out how best to normalize this sort of data. The exosomes were isolated from tissue culture medium, and reading the literature I can find spiked in external controls as well as miRNAs used as endogenous controls. The issue is that exosomes are packaged selectively (we think) and I don't suppose I can assume that there are specific ones (for example let7) which are equally loaded in all cell types I'm screening. Is there a consensus in this matter?
Instead of normalising, why not do an absolute quantitation? You can buy phosphorylated RNA oligos which will work very well in taqman assays. Prepare 10-fold dilutions of the oligos and RT then PCR them in the normal way. You get great standard curves and you can establish the copy number of miRNAs in your exomes. Happy to pass on my protocol if you'd like?
I don't think there is a consensus and that's one of the things that makes exosomes interesting. But… what if… you take a couple reference miRNA as endogenous controls, and on the side you check the variability of those in treated versus control, normalizing to total amount of miRNA for instance… Just thinking out loud… What do you think?
That's what I thought... trying multiple "reference" miRNAs, and see what happens... :)
No consensus *sigh*, and I wish the general concept of "reference miRNAs" was fully resolved by itself, let alone in exosome context! For instance, Northern blots are normalised to total RNA load or rRNA staining - who said that RNA Pol I activity remains stable under experimental manipulation? Similarly, people use various snRNAs or tRNAs as references for miRNAs - Pol III is definitely affected by cell growth rates, so not much of a biologically-valid assumption there either.
Think carefully about the biological question you are trying to pose and the experimental contrasts you are making.
Selective packaging? You will need data about many miRNAs (ideally, microarray or sequencing) followed by ranking or fold-enrichment analysis. No single miRNA will serve as a reference.
Efficiency of secretion? You will need spike ins to check extraction and RT-qPCR efficiency. C. elegans or plant synthetic mimics added to medium and cell lysates prior to extraction will allow you to normalise amount secreted to amount in cells.
There are other combinations, but the reference should be chosen according to YOUR biological question, not according to someone else's consistently reported bad reference :-)
Will be happy to offer ideas if you post further experimental details.
Good luck!
Dear Anna,
Thank you for the answer. These are precisely my problems right now. The main question we have is the presence of viral miRNA in exosomes shed by infected cells -this should be easy to answer (yes/no), and even if the normalization is not perfect. However, if I get into the whole issue of comparing miRNA profiles of mock infected and infected cells, I enter into the problematic territory. We have a limited number of miRNAs we can use, as sequencing is the next step for the future (normalization of THAT data will be an issue as well, but I can just tell the bioinformatics people to deal with it, thankfully...). Perhaps this really should be left for sequencing instead of qPCR.
I was thinking of spiking the sample with some mimics before the RT step, but I'm pretty sure it's not a catch-all for the issues. The problem is I'm only now learning qPCR, so I don't have a great deal of experience. If you have any suggestions I would be grateful to hear them. Thank you very much.
I would suggest a high-throughput screening of exosomal miRNAs of your different cell types and normalizing on mean expression value as published by Mestdagh et al.. I have attached the link in case you're interested.
http://genomebiology.com/2009/10/6/r64
Instead of normalising, why not do an absolute quantitation? You can buy phosphorylated RNA oligos which will work very well in taqman assays. Prepare 10-fold dilutions of the oligos and RT then PCR them in the normal way. You get great standard curves and you can establish the copy number of miRNAs in your exomes. Happy to pass on my protocol if you'd like?
I agree with Dr. Jan Van Deun's suggestion, normalizing on global mean expression value.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA