I have to design primers based on stem-loop primer method for different miRNAs as I am interested to check their expression in my clinical samples. Can anybody please tell me which miRNA sequence (-5p or -3p) to choose when designing primers against it for expression profiling? For instance, based on several serum profiling studies, I am looking to use miR-16 as endogenous control. But I am confused to select which miR-16 sequence for primer design following recent updates of mirBase? -5p or -3p because the previous published data just lists miR-16. Same is the case for miR-21, -221 etc whose expression I want to check in my samples. Please guide.
Reading the following article in mirbase, helped me put a similar doubt to rest.
http://www.mirbase.org/blog/2011/04/whats-in-a-name/
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.
@sneha: Thats right but I have to design primers by myself due to cost issues and etc so the question is which mature miRNA sequence to select for this purpose, -5p or 3p?
I think you should try the one with high profiling, which is always the functional strand and.
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)
and...
Good luck!
Try to use RNU6B as endogenous control for miRNA expression analaysis. http://bioinfo.invitrogen.com/genome-database/browse/mirna/keyword/RNU6b?ICID=uc-mirna-RNU6b
I have used this and working well.
3p and 5p denote two mature miRNas originating from the same precursor, so I would first check whether in miRNA expression atlas both have been detected in your cell system http://www.microrna.org/microrna/getExprForm.do Or only one of them, then design your primers accordingly.
And for sure I would not use a miRNA as 'housekeeping gene', use as recommended some other sncRNA ie. RNU6B, RNU58B... and to be completely safe, if you intend to do real time PCR, then I would choose at least two housekeeping genes.
Good luck!!
They are two different microRNA's, you need to figure out which one is important to your studies or experiments.
Try using TaqMan probes or miScript PCR system, they have customized primers for your MIRNA of interest. I have used miScript for my work., using RNU6B and Actin as internal control. They are just fine!
you can check miRs previous ID on miRBase, so you can find out miR-16 as an internal control refere to which ones ,3p or 5p. also check out deep sequencing reads on miRBase to identify which one is more stable and more frequent.
Reading the following article in mirbase, helped me put a similar doubt to rest.
http://www.mirbase.org/blog/2011/04/whats-in-a-name/
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.
I've asked this on another related thread but there are more people posting here so I might have more chance of a reply:
Why do some miRNAs not have the 3p or 5p annotation? For example, hsa-miR-3687? It's entry in the mirBase can be found here:
http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0016088
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