I’m doing qPCR for evaluating the resistance gene expression in pseudomonas aeruginosa, but melting peaks are not specific. I tried different annealing times and temperatures, different concentrations of cDNA and primer, and even used touchdown qPCR (55- 65 ). I tried with another set of primers but the results didn’t change. These new primers show good results with PAO1 (standard strain) but in the same condition, the clinical species melt peaks aren’t specific except for 16S rRNA which has a good peak in every condition. By the way, I confirmed the bacterial isolate by tox A and rpo D which are specific for the Pseudomonas aeruginosa. The primers are specific based on the blast results. (I used syberGreen)
The melting peak is the LEAST useful part of qPCR (and often not even included in publications). The important data are the Ct values if you are measuring gene expression.
What size are your amplicons? I can tell you right now that products that are either larger than ~100 bp or have regions with very different GC% will melt in "messy"/multi-peak curves.
Can you upload a picture of the peaks? That would be very helpful. A small "shoulder" region is typical but a very broad or multiple peaks are problematic. What happens if you use these primers for standard end-point PCR? Do you get a single crisp band?
Amy Klocko
primers have single bands on gel and one peak in qPCR for standard strain. I get different peaks in each reaction for the same gene.
Ah, I see what you mean about the double peaks. Are your target genes part of the core genome or located on plasmids? It may be possible there is genetic variation present in your targets.
Another possibility is the size and GC composition of your target fragments. Generally, smaller is better for QPCR, and evenly distributed %GC helps with single peak formation.
My vote would be to try a different set or two of primers for each target. I used to design 3 sets per target, test them all (melting curve and efficiency), and select the 1 best set for use.
Hi there. I think you should try different pairs of primers and use the primers design tool to avoid dimer or other problems. It's also possible that there're unspecific amplification. Also how's the quality of your template? Check if your cDNA or RNA quality is good enough to do qPCR. My experience is that some clinical strains can overproduce extrapolysaccharids that may contaminate final products during RNA extraction.
Amy Klocko
my target genes are part of score genome. results are similar for two sets of primer
Jiang Yujun
the quality of cDNA and RNA was good based on the nanodrop results
The only way to know for sure what is being amplified is to sequence the products. My bet is that you have regions of variable %GC in your samples from the clinical specimens that is not present in the standard strain.
Again, if you are measuring gene expression, you should be more concerned about the Ct values. And if your primer efficiency is 95-105%.
As a note, you'll need to sub-clone your products for sequencing and use primers located in the vector backbone. The products are very small and sequencing data only starts ~20 bp after the primer ends so if you send the products for direct sequencing you may not get enough/all the data.
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