Stem-loop RT primers: how to anneal? - (Feb/13/2008 )
We have just started working on microRNA profiling in melanomas and are checking expression levels of a few miRNAs. We are using the stem-loop and qPCR primers from Ambion but they are so expensive that we cannot work with larger numbers of miRNAs.
I have read pretty much all the papers concerning the stem-loop RT primers and I know that people are starting synthesizing their own (like I want to do) but none of the papers has any details about the annealing conditions for such primers – temperatures, times etc.
And also how to check whether the major entities in the tube are the stem loops and not primer-dimers..
If anybody has any idea on how to anneal and create stem-loop primers, I would be grateful if he/she could share it with me…not to mention how extremely grateful our budget would be!!!
Thanx a lot in advance!!!
i haven't exactly worked with miRNA... but i did do some PCR with primers that had to anneal to a stem-loop region.
what you need to do is a gradient PCR. they usually need higher temps in order to not produce a mess. (i used 56-65 'C gradient, and found that 62 worked well for me)
also, they don't need much time. i used maybe 15-30 second annealing time. but this will depend on your polymerase.
to create them, i just looked at what i had to PCR up... and then double checked the stuff with primer3 (use google to find this program).