Hello!
We have recently initiated several projects focused on analyzing microRNA (miRNA) expression. Although there are numerous resources available on quantifying miRNA expression using qPCR, we are currently facing some challenges in obtaining consistent and satisfactory results, particularly in the following areas:
We would greatly appreciate any guidance, recommendations, or resources you could share to help address these issues.
Thank you in advance for your support.
Ahmed
Those are a LOT of complications. Which one do you want to try to fix first?
Do a literature search for the primers. And read about qPCR experimental design on company websites for folks who sell the machines https://www.thermofisher.com/blog/behindthebench/ten-tips-for-successful-qpcr/
Efficient extraction means that you need to have lots of fresh (or immediately flash frozen) starting material. Read the protocol to see what minimum and maximum amounts you can have. If it's a fibrous/oily/chitin/other hard to grind sample, you'll need to adapt the protocol.
If your miRNA of interest is low-level or rarely expressed, then you're going to see high Ct values even if the extraction yield is high.
Good luck!
Primer-wise, there are tons of pre-designed primers available for taq if you know your target(s), depending on how you are going about your studies. Personally, I'd start there.
What type of samples (culture/tissue/supernatant/blood/EVs/etc) are you trying to get miRNA from? That will entirely determine the method that would be best for extraction
Literature can tell you what a good "reference" miR would be for your sample type. That can be how you estimate whether your got accurate measurements of expression levels. I see miR-16-5p as a common reference. It is not incredibly abundant, but consistent (relatively) between people.
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