I've never done RT PCR assays before. I'm an independent researcher and am worried that multiplex assays are more expensive than singleplex. However, I'm not sure singleplex assays will give similar results as multiplex assays.
singleplex pcr give similar results to some extent, but there we can't find out the pipetting errors made by us so we choose multiplex pcr. Multiplex is better option but if you can'nt affort it you can reduce the reaction amount to 12.5micro lit. But make sure u are using reactions in triplicates.
also confirm the minimum amount of reaction your RT PCR machine can hold. Dont use the reaction mixture less than that
Thank you nidhi for your kind reply. I believe that the primers need to have similar melting points for multiplex reactions?
In Real time PCRs Multiples is not recommended if you are doing quantification or semi quantification. its ok for SNPs.
Why do u want multiplex control and gene of interest? Once u standardize housekeeping gene (control) u can do n number of reactions for gene of interest
@chakradhar It was suggested in some literatures that it can reduce pipetting errors. Im pretty much worried about the price though. THank you so much for your helpful answers people!
Hi you'll have to do a lot of optimization if you multiplexing (I assume you are using TaqMan probes and primers). Better to do a singleplex reaction which has ROX as added as an internal control. This can take care of your pipetting errors, if any. I assume this is your main concern.
Also you would be using an endogenous control like GAPDH/beta actin etc. so you can compare expression across samples
Hope that helps
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