Traditionally, 10x cDNA dilution works the best. Could you please explain what does "multiplex mix of fungal specific primers" means? Does it means each sample was mixed with multiple primer pairs specific to fungus!

In general, if the Ct value is not specific for similarly diluted samples coming from the same treatment that would mean the primers or the qPCR conditions are not optimized. First, you can start optimizing the Tm for qPCR. If it doesn't help, you can check if the primers are making any secondary structures that may cause problems in the efficient amplification of the samples. Designing new primers would be better then.

Dear Megan,

I propose you using MLPA version of multiplex PCR, since it needs only one set of universal primers to amplify up to 50 different sequences. However, I guess you should design and order some probes, but after that, it will make your procedure much easier. I also recommend you using a software such as LinReg, instead of using standard curve. Since the standard curve tells you about the efficiency of the primers, but the software tells you the efficiency of each reaction within each well. I hope you find my answer useful. Regards

Hi,

I guess a multiplex qPCR is difficult to set up as all involved amplicons can have different rates of amplification and there can be competition between them.

You can pick the content of some wells in the plate and run them on a 2.5 - 3 % agarose gel to see what the amplicons look like.

How many targets are there in the multiplex? Have all the probes labeled with the same dye sounds strange to me, I'd rather expect to do it with 1 dye per target and just run them in 1 single reaction rather than separate reactions to save limited material. But this is a naive remark, I've never performed a multiplex qPCR.

Regarding this issue:

"My amplification plots look good and how you would expect them to, however the curves are all over the place."

I'd say that at a given amount of starting cDNA template material you can get a nice amplification curve that is the sum of all participating amplicons, but this repartition of contribution to give the final detected ROX signal does not evolve linearly with serial dilution of the concentration of the starting template for each of them and gives a strange standard curve. If that is the case, the assay is rather qualitative and binary and test samples considered positive or negative above and below a certain threshold but without quantification.

Regarding the standards dilution range, as Sayante Bera says, I also prefer 10x serial dilutions (i do 10e0 to 10e-5) as it covers a larger CT range (log 2(10e5) = 16,6 CT). I've seen people do 1/2, 1/5, 1/125 and 1/625 standards (log2(625 = 9,3 CT). However this is not a problem if the tested samples all end up nicely somewhere within the standards.

Finally, a possible alternative to multiplexing is the fluidigm technology that probably requires less cDNA for x reactions than the usual plates and finally costs less than setting up a multiplex

Hope this helps

Best,

Daniel