In an effort to develop a triplex probe qPCR I started uniplex qPCR with SYBR green. For two targets the test performed well. For the 3rd target I got late Ct values several times. Primers, probe were designed to produce amplicon size of 100 bp, but were degenerates (targeting Papilloma virus L1 gene). Thermal profile modified to have 3 steps; Segment 1;10 m at 95 C, segment 2: 95 for 20 s,53 for 30 s (40 cycles) and segment 3 95 for 1 m, 50 for 30s and 95 for 30s.
Hi, Abdelmalik, you may have a very low level of cDNA of this gene. You can try to increase cDNA concentration to get better ct value.
It is not clear from your information if you are amplifying target or not. The late Cq you are seeing could be due to low abundance of your target, or it could also be due to detection of primer dimers rather than your intended target, Did your melt curve show a specific product? if so, what was the melting temperature? You could also confirm whether you have dimer or specific product using electrophoresis.
If the amplification turns out to be only primer dimers, then you may need to optimise or redesign the assay.If you are getting both product and dimer, you can add more template as Taras suggested, or carrier (non target) DNA to solve the problem, as these are more likely to form when there is low amounts of DNA in the reaction.
Your cycling protocol is also unusual- the very low annealing/extension temperature could exacerbate the non specific amplification, as well as being sub optimal for Polymerase activity. If you are using Taq, then I would recommend a separate 72 C extension step to maximise your product yield.
Many thanks for the answers, dear Scientists. Taras, the template I tested is a plasmid containing the target insert and the concentration was high enough (50 pg). Gerwyn, Yes, I am thinking of primers dimer product, but the dissociation curve was good, only one peak. Your proposal to mofify the cycling program is valid and I will do it next time.
Dr. Khalafalla,
I agree with Gerwyn that your protocol is unusual and support the notion that your annealing step is a problem.
Here is what you will want to do:
1) Begin your entire process using basic PCR parameters.
Initial Denaturation: 95 C, 5 min
Denaturation: 95 C, 45 sec
Annealing: 65 C, 20 sec
Extension: 72 C, 45 sec
(40 cycles)
Melting Curve (Your dissociation method seems fine for now)
2) Run a gradient-style assay for each target.
One reaction will consist of a 54 C annealing, the next will consist of a 56 C, 58 C, 60 C, 62 C, 64 C, 66 C, 68 C, and 70 C.
3) Compare the Ct values between all three targets at the various temperatures described above and determine which temperature will allow the multiple-target reaction to simultaneously amplify your target in harmony.
It is a time and material consuming practice of optimization. However, given the goals of your study it is necessary and will ultimately pay off in developing a multiplex reaction if you are struggling with one of your targets keeping up in reaction.
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