Question 2 of 9 Intermediate Troubleshooting
What could cause amplification in a qPCR no-template control?
Short answer
An amplifying qPCR no-template control may contain contaminating target DNA or amplified product, or it may reflect primer-dimer formation. The right response is to inspect the curves and controls, investigate where the signal came from and correct the underlying issue before trusting affected results.
Why
Do not treat every late NTC fluorescence trace as equivalent. First examine the assay chemistry, the shape of the amplification curve and, with intercalating dyes, the melt profile. A melt peak at a temperature different from the intended product can suggest primer-dimers, whereas a peak matching the intended amplicon may raise concern about contamination.
Review reagent preparation, plate setup, pipette use, aerosol carryover and separation between pre-PCR and post-PCR areas. If contamination is suspected, repeat with fresh reagents and appropriate blanks. If primer-dimers are suspected, reassess primer concentration, complementarity and annealing conditions.
When evaluating consequences, compare the NTC signal with low-level samples and the validated detection range. A clean rerun does not retroactively validate a failed previous run.
A strong answer covers
- Separate plausible causes and plan discriminating tests