multiplex rt pcr - (Feb/07/2007 )

hi everyone,

i'm just a beginner with the multiplex pcr, i'm trying to amplify 19 different fragments with 40 individual samples, i found an article by invitrogen for their product called accuprime which says it can specifically amplify like 10 or more sets of primers. i tried using the one step protocol with 2 sets and the optimal cycles appear to be 30, at 36 it gets saturated, i did that by semiquantitative pcr.
does anyone know if it's best to do the one step rt pcr with different sets of primers or doing the cDNA first and then do the multiplex pcr? thanks a lot for your help!

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with multpilexing you need to optimise the conditions for each primer set individually before throwing them altogether. Furthermore, complications can arise from differing amplification efficencies from your different loci. To do what you want to, I would say it would be better to look at your samples individually if you have enough to do so,

otherwise it would be a night mare to optimise.

Nick

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hi nick, thanks for your advise, when i designed the primers i tried to give them as similar Tm and GC content as possible. the main problem we have here is the budget, we have to reduce the number of reactions since it'd be around 780 if i do them individually. one of the main problems i've seen is that there's preference for the smaller fragments than the larger ones, but then, if i use the same combination of primers for each of the different samples, let's say primer a, b, c and d with the 40 samples, the variation in the amplification of the fragments among samples should be the same, right?
once again, thanks for your replies. have a nice day.

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Hi,

I'm trying to make a multiplex PCR with primers i have already designed and i want to find an online software wich permits me to predict if i have any interaction between my different primers and probes.

Thanks for your reply.

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@rodpck,

you are abosulutely right if the assay is 100% efficent, murphy's law states otherwise

good luck with it.

Nick

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damn murphy! and damn company which was supposed to deliver my enzyme last week and hasn't come yet ! i'll let you know if it worked or it was a complete mess, thanks again.

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