I am using genomic DNA from sperm as the template, try to do sex-ratio relative quantification, when I ran the qPCR, it shows the shoulder in the melt curve, When I ran a gel for the qPCR products, it shows one band, but above the bands, there are some none specific thing shown up.
I am using a autosomal primer for reference gene, but either using autosomal primer or GAPDH, it gives me the shoulder in the melt curve.
1) the primers are optimized for this PCR 2) there is no primer dimers.
Can this be a problem when running the gDNA as template? Or using ROX and SYBR will give the problem?
Is this a good way to do the sex-ratio determination? When I did the calibration curve, more input of my template, did not increase the CT value (improve the CT value).
You can get a "shouldered peak" from a single PCR product if it has regions of different GC% content. AT rich areas melt at a lower temp than GC rich areas. It's totally normal. You can try to minimize the shoulder by redesigning primers to create shorter products, but you don't need to.
As long as your controls are working well, you've confirmed the presence of a single product by gel electrophoresis, and your primer efficiency is 95-105%, then you're all set!
Good luck with your experiments!
"the primers are optimized for this PCR" did you mean concentration or efficiency? or both?
I would recommend triplicate experiment for more reliable data since you have technical errors (Ct difference is more than 0,5).
Since Applied Biosystem 7500 device uses halogen bulbs, so yes you need to use ROX as normaliser for bacground noise. Moreover, keep your device calibrated.
Never devices have LEDs, so there is no calibration needed and no shifting on illumination.
Good luck!
Cheers
Please see my melting curve plot for SSR marker. I have "shoulders" since that primer amplified more than one fragments for some genotypes.
Sincerely
Thanks for your answer Kaan Hürkan Katie A Burnette . My previous gel pic for checking the products, please see attached. It's possible amplify more than one fragment since i am using the genomic DNA. The annealing Tm is optimized for this PCR, but for the concentration, we are trying to do a concentration dose curve to see which work the best. Also, I am thinking that it can be the primers issue, the efficiency.
Our ABI 7500 haven't been calibrated, maybe for a long time. But I don't think this may cause the problem, because the machine also work for other people.
Sincerely,
Delong
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