Hi,

if you know your TOI is expressed 10ng cDNA is sufficient. In addition for many qPCR mixes you can go with a standard temperature protocol and first focus on performcance. Also for formulations I would suggest to first start with the proposed concentration for Primer and probes as indicated in the handbooks

Good luck

Sven

My usual approach is one of "primers are cheap, and a robust assay is far better than a fussy assay": in other words, if your assay NEEDS careful optimisation, it's cheaper and easier to just redesign your primers until you get an assay that's basically bomb-proof. I typically order two or three pairs of primers to my target transcript, use the same generic cycling conditions I use for all my other qPCRs, and then just keep the one that works. Usually they all work acceptably, in which case I'll pick the one that looks nicest/cleanest.

Optimization first .

For my experience Optimization before essay already give the suitest performance.

Good luck

Hi all,

Thanks for the guidance.

May I know in order to carry out assay optimization, do I need to do it only on housekeeping gene or I need to do both on my housekeeping gene and targeted gene?

Thanks in advance.

Greetings,

The optimization has to be done for every single paro of primers, being those your target gene or HK genes, you are planning to evaluate expression stability. Once you manage to get your Rx conditions optimized, perform serial dilutions, either for your previous amplicons or cDNA, and perform STD. Curves. For the latter, the best way to get a Good R^2 is to use a vortex to properly mix everything during 15 seconds each round.