HELP! early ntc amplification, reaches quickly plateau

Go to the profile of matilde canedo
Feb 21, 2018
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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.

2 Comments

Go to the profile of Roberto Rosati
Roberto Rosati 7 months ago

Hi,
would you think that this could be due to incorrect settings for baseline and threshold? That'd be my first guess.

Go to the profile of OldCloner
OldCloner 7 months ago

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!