miRNA real-time using Sybr green - (Jun/30/2008 )
I experience some problem with the real-time PCR quantification of miRNAs. I follow the protocol Biotechniques 2005. I always get a second peak in my dissosiation curve for all different miRNAs I have tested so far (see images). I use an ABI 7500 with the sybr green from ABI. PolyA tailing was done with Ambions kit of either total RNA or enriched small RNAs. The second peak seems to depend on the specific primer concentration. I have already extended the primers by two A's and get the same results. I would appreciate any suggestions how to solve the problem.
Are you using the Ambion mirVana qRT-PCR kit? I had really, really bad experiences with this kit. To be quite blunt, I wouldn't even try troubleshooting if you're using this kit, go straight to the ABI TaqMan primer/probe sets instead which works really well. If you're not using this kit, where did you get your primers from?
But seriously, it was useless in my hands and I wasted a good 3 or 4 months on it. Even the Ambion tech support people couldn't offer much help or didn't even seem to have the opinion that it could work!!!
No I don't use this kit, only the polyA tailing kit from them. I don't know wether the problem lies with the polyA. But obviously I get products so I assume the tailing and the cDNA synthesis is fine. The adapter and antisense primer I use are from the paper (Biotechniques, 2005). The specific sense primer is from the mature miRNA sequence plus two A's at the 3'-end. The cDNA synthesis is done using the ABI kit. Until now the SybrGreen worked well with other targets (except miRNA). I played already with the idea using LNA. But it would be nice to have an idea whats going on and what second product I get, because the NTCs don't show any product, so the primers alone are fine.
Have you tried running your product on a gel? Could you be amplifying mature and pre-miRNA? Could you also check by in silico PCR at http://genome.ucsc.edu/cgi-bin/hgPcr , and see how many products are predicted?
Yes I have run the real-time reaction in an agarose gel, but only for the 50/50 nM reaction. I see only one band around the size I would expect. Because the first peak has a lower melting temperature I would not think that it is the pre-mature miRNA. The second peak has the right melting temp. according to the theoretical calculations.
The in silicio does not work because the adapter primer is arteficial and has nothing to do with the genomic DNA.
Thanks for the recommendations so far.
Sorry, I don't know what else to suggest. I hope that you're able to work it out.
Do you use purified small molecular RNAs or total RNA as template? I had similar problem when I used purified small molecular RNAs instead of total RNA.
Actually I have used both, starting with total RNA and used later purified miRNA. The same results. Lately I used a different adapter primer and got an increase in the product with the lower melting temp.
Didn't solve the problem yet.