I am planning to detect expression of miRNAs in human tissues. Could anyone please let me know how to design primers/any online resources to design primers for miRNAs? Is it similar as we design primer for any cellular gene?
Identical information provided in the 1st reply by Budak Melayu is available at the "Stratagene’s High-Specificity Primer Design Guidelines" PDF (link below).
This is an excerpt of this excellent explanation by Sam, 2011.
The name of a miRNA contains some human-readable information. If you stop reading this post halfway, you’ll likely think this is a good thing. Which of course it is, as long as we recognise the limitations. Hold on to the end and hopefully you’ll see that names can create some issues.
Take for example, hsa-mir-20b. The “hsa” tells us it is a human miRNA. The “20″ tells us that was discovered early — it’s only the 20th family that was named. “20b” tells us that it is related to another miRNA that we can guess is probably called hsa-mir-20a. We can go further — the (lack of) capitalisation of “mir” tells us we’re talking about the miRNA precursor. Or maybe the genomic locus, or maybe the primary transcript, or maybe the extended hairpin that includes the precursor. So that’s already less useful.
hsa-mir-20b has two mature products, named hsa-miR-20b and hsa-miR-20b* (as of this moment — as you’ll see below, this will change). “miR” tells us we’re talking about a mature sequence. In this case miR-20b arises from the 5′ arm of the mir-20b hairpin, and miR-20b* arises from the 3′ arm. The “*” tells us that miR-20b* is considered a “minor” product. That means miR-20b* is found in the cell at lower concentration than miR-20b. It is often inferred that miR-20b* is non-functional, and you’ve probably noticed that miR* sequences in general magically disappear in most pictures of miRNA biogenesis, while the dominant arm is magically incorporated into the RISC complex.
If you are checking the expression of the mature miRNA, and you know the sequence of the mature miRNA then use the seqence(s) of the mature miRNA, regardless of 5p or 3p, if both express in you tissue then you shold (try) to detect all of them. Also a possibility , if you dont care about the 3/5p and there is a possibility, try to choose the primers that anneal on both (in some cases the mature miRNA sequence is very similar). I would suggest, to first figure out which specific mature form is relevant for you (literature, expression studies) and then focus on them. Or you can first pick the most interesting ones and then the rest, because i would suspect it will be quite difficult to properly conduct expression study on the several miRNA with 'home-made' primers.
At the end it might be cheaper to order pre-prepared probes for expression assays, because you don't need much optimization (in most of the cases)
Identical information provided in the 1st reply by Budak Melayu is available at the "Stratagene’s High-Specificity Primer Design Guidelines" PDF (link below).
Saied Afshar, you can use good quality mirna based product such as EXIQON miRCURY LNA™ Universal RT microRNA PCR and convert your all mirna from total rna to cDNA.
@ Deilson Elgui de Oliveirae I am trying to get an access to the link that you have mentioned above, but it is not opening. It is therefore requested to please send the protocol for miRNA primer designing. I will be grateful for your this act of kindness.