i run qpcr for gen ki67 with reference gene beta actin. before do qpcr., I already optimized the annealing temperature in pcr. in gel electrophoresis there is no indication of dimer. but unfortunately, in qpcr there is indication the primer got dimer and has multiple peak. could you help me how to fix this problem? i already used the low concetration 0.25 uM for the primer.
Dear Swara,
Seeing only the image, its seem a clearly effect of primer-dimer, or contaminants in the qPCR reaction. You can know easily what is the problem looking the melt curves of the negative controls.
Good luck!
You may not see dimers on your gel because they are short and bind less ethidium bromide than amplicons do, so they are less bright. And ethidium bromide moves in the gel in the opposite direction compared to DNA, so if you run your gel a little too long your shorter bands become colorless. You can avoid it if you add ethidium bromide to electrophoresis buffer as well.
Are these melt curves from replicas of the same reaction? What are Ct for these samples? Do you see such melt curves only in particular sample, or in all samples? Did you check melt curves or run gel electrophoresis before when you optimized primer annealing temperature, was it looking like you have a single product?
If you see this problem in all samples, you should redesign primers.
If you didn't see this problem when you did primer Tm optimization with positive control sample, but see the problem with all test samples, something was wrong with preparation of all your test samples, or your control was not appropriate.
If you only have this problem with one particular sample, it may be contaminated and you see byproducts amplified by false priming. Or, if this sample has really high Ct, more than 30, you may have too little template in it and the sample behaves like no-template control. No-template controls can have really weird (and different!) melt curves because even the most specific primers usually start to form dimers or multimers or something after 32+ cycles of amplification.
Looking at the melt curve profile, it seems that the amplicon size is greater 200 bases. In such cases when size of amplicon is higher you may see multiple peaks that are because of either non-specific amplifications or some kind of contamination which otherwise is not visible on gel. The primer dimer melt curve Tm will be typically below 80 degree C due to smaller size of dimers.
- Badges
- Science method