Dear Hussein,
You can get predesigned form of interested miRNA primers sequences in miRBASE database. Nowadays most of companies directly sells pre-mix(FP+RP) pre-designed primer of most of the miRNAs.
Yes you can for some microRNA. Problem with SYBR green asssay is that you can't discriminate between closely related families i.e. let-7 and you will detect the pre-microRNA transcript. Here's what i did
1) Found the abundance of my microRNA of interest from NGS data in my tissue. You won't be able accurately quantify transcripts that are >30Cts. I picked transcripts that are >100 FPKMs.
2) Checked the miRBase for mature microRNA sequence and designed oligos for it using the NCode cDNA kit's recommendations. Can't find the manual with this information online, not sure if Thermo updated it but you can ask them.
3) Out of the dozen primer i designed, only about 2 were not usable. Not bad given how cheap they are vs. using Taqman.
- Badges
- Science method