HELP! early ntc amplification, reaches quickly plateau

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HELP! early ntc amplification, reaches quickly plateau

Postby Matilde Canedo » Feb 21 2018 6:59 pm

I'm new to qPCR, and I’ve been testing the AR expression on canine tissues. I’m currently using a pair of primers that have been tested in other article for canines.The issue I’m having is that I have very early amplification of ntc AR (before the amplification of cDNA templates) but also this amplification curve looks weird since it reaches quickly the plateau phase (flattened). This problem persisted even after changing to a much more specific cycling. If anyone has any tips, or ideas on how I may be able to explain this, I’ll be very gratefull.
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Re: HELP! early ntc amplification, reaches quickly plateau

Postby r.rosati » Feb 22 2018 3:06 pm

Hi,
would you think that this could be due to incorrect settings for baseline and threshold? That'd be my first guess.
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Re: HELP! early ntc amplification, reaches quickly plateau

Postby 29yrsExperience » Apr 06 2018 8:45 pm

Are you using Sybr green (my guess) or TaqMan probes? If it is Sybr, what do your melt curves for ntc vs. positive control look like? A good amp of a positive sample should give a clean single peak of a specific fairly high temperature. Primer-dimers give weird low temp. melt curve peaks. Have you tried running some of your amplified products including the ntc on a gel to see whether you are getting primer-dimer or the expected size amplicon in your ntc? (Do this away from where you set up PCR reactions).

Also, in my experience, sometimes primer-dimer can be reduced or eliminated by simply cutting back the amount of primer in the reaction. You can cut primer concentration in half without preventing true amplification of a positive sample.
Good luck!
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