Hi!
Does anyone know of a published RT-qPCR primer design?
I have found different platforms that I could use to design my primers but not a step by step protocol...
Thank you!
There isn't a step-by-step protocol because everyone's project is a bit different. I suggest you chat with folks who do qPCR and come up with a plan that is custom to your research.
As a general rule, your primers need to anneal and amplify from exon regions only. Ideally, the primers "span" an exon-exon junction so you can't amplify from gDNA. Or at least have an intron between them so the gDNA product is obviously larger.
You also want primers that create small products (~100-120bp), are specific to your target gene/locus, and have high efficiency at the annealing temp for your qPCR kit.
Most primer pairs won't work, you'll have to test out several. Don't trust anything that you haven't tested yourself - I don't care if the primers are published/recommended by a colleague/etc.
And you'll need to decide on your controls NOW.
And clone your region of interest into a plasmid to use in your standard curve [or make a LOT of cDNA from a source/tissue/treatment that you know expresses the gene(s)]
Good luck!
You can design basically as you are used to using the tips Katie gave to you above, and also when you design your primers you can go to IDT website to analyze them. There you can check their DELTA G, if that primers can form hairpin, what would be the Tm of the primers, and etc. It is good if you can design pair of primers with similar Tm.
Go to https://www.idtdna.com/pages, in TOOLS choose "OligoAnalyzer tool".
Beatriz Cristina Dias Oliveira Obrigada!!
I was able to do it just using the nbci website! Had everything included :)
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