I designed Primers for miRNAs using mirPrimer algorithm. However when l analysed them by Oligoanalyzer 3.1, it shows Primer-dimer and secondary structure formation. I request your suggestion regarding the same.
hi Javaid Wani
miRNA primer has universal sequences. The miRDB database provides stem-loop & mature miRNA sequence. Prefer predesigned primers of your interested miRNA from the standard company. Nowadays, Companies do a slight modification or say a technology, which enhances the stability and avoid unwanted binding. Qiagen has LNA tech. based miRNA primers, it has readymade, pre-mix(FP+RP) pre-designed primer of most of the miRNAs.Just dissolve and use 1ul for per rxn.
Regards
Saurabh
Hi
There is a webtool called sRNAPrimerDB is available for miRNA primer designing as well as it has a list of predesigned primers. The primer or probe design tool specifically for small non-coding RNAs (sncRNAs), such as microRNA (miRNA, 20-25 nts), PIWI-interacting RNA (piRNA, 24-32 nts), short interfering RNA (siRNA, 20-25 nts). Hope this may be useful for your research.
all the best
The rules for 'good' primer design don't always apply well to small non-coding RNAs. With most routine PCR and qPCR, if a bit of sequence is ugly (high GC, low GC, hairpin, homopolymer, etc) you can move your primers somewhere else so that you can design them nicely. With small RNAs that are barely (if at all) larger than a primer, you kind of just have to use the sequence you have, and hope that it works. Optimising qPCR parameters (annealing temperature especially) can often help a lot, but sometimes your best option will be inserting an LNA base or similar to increase the annealing temp of the primer. The tools others have suggested are good places to start. I would also advise that in this specific case, don't be afraid to order a primer and test it even if it breaks the rules for 'good' primer design because a lot of mine worked anyway :)
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