TaqMan single v duplexing - (Apr/01/2008 )
Please can anyone help! I am trying to look at both my target gene and endogenous control (18S) in the same reaction (duplexing). I have designed custom TaqMan primers and probes, and am currently trying to optimise them for duplexing.
When I duplex them, the CT values for 18S and target go up by about 2 to 3. This happens at whichever different concentrations of primers I choose to use (ranging from 40 to 900nM). Please can anyone have any ideas as to what may be causing this delayed CT values?
MAny thanks in advance
If you run them together it is logical that Ct values would be higher, because they compete for the reaction components. What exactly are your Ct values?
The Ct values for 18S are around 15 (singleplex) and 18/19 (duplex); target gene is 23 (singleplex) and 26 (duplex). I'm currently using a qPCR mastermix supplied by ABI; they dont reveal how much magnesium or Taq there is in the mix so I'm not sure how to go about increasing reaction components. Do you recomment any other supplier?
Thank you so much for your reply
As you can see, the differences is always about 3 Ct. You can't (and shouldn't IMO) do anything with the mix, but try using a lower concentration of your 18S primers. Housekeeping should be around same Ct as target, especially in duplex.
Ok I have given it a go.. I did a series of successive dilutions of the 18S primers, and still got delayed CTs for both genes, up to the point at which the 18S primers were so low, that I didnt get any PCR. I'm not sure what to do next! Please help!
-Trof is rigth. That's why I never use 18S as reference but HPRT, ACTB, PGK1, PBGD, ...
-Maybe you try another master which is optimized for duplexing - I can recommend you the FastStart universal probe master (rox) from Roche. I work with it on the abi machines and for me it works better than those of ABI.
-Sometimes it helps to prolong the elongation step up to 90 seconds...