Hey everyone. I have a question.
I'm doing the in vitro silencing of a small RNA of 79 bp (a snoRNA specifically), and I need to design a protocol for the detection of the known-down of this RNA. I would like to perform a real time RT-PCR with intercalating dyes (like SYBR) but I can't design primers against any region because the target have too low GC content. I read about some hairpin primers, but I would like to know if you have some experience with this.
Thank you very much!
Aldo.
Thank you Ali! I designed this pair of primers, which are like the best option according the Tm and lenght:
F: TGCAGATGATGTAAAAGAATATTTGC (26 bp, Tm 52.5, GC 30,8%)
R: CAGCGATCTTGGTGGTTTA (19 bp, Tm 52.5, GC 47,4%)
The problem in the GC% is for the forward primer. The melting temperature of both still low (52,5)... may these primers work at the annealing temperature of 50°C?
Thanks!
There's no problem for the reverse primer, but adding or subtracting bases for the forward only decreases the Tm, the maximum that I could obtain is 52,5. This is the template:
TGCAGATGATGTAAAAGAATATTTGCTATCTGAGAGATGGTGATGACATTTTAAACCACCAAGATCGCTGATGCA
Would be too risky to try primers with that Tm? should I concider another approach?
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA