LimsPulse
Bioanalytical

Real-time PCR (qPCR) system

A thermal cycler with a built-in fluorescence reader. It copies a chosen DNA sequence cycle by cycle and measures the fluorescence in every well after each cycle, so the starting amount of DNA can be calculated from a standard curve.

  • Biotechnology
  • Biopharmaceutical
  • Cell and gene therapy
  • Academic research
  • Agricultural biotech
  • Food authenticity
Photo of a Real-time PCR (qPCR) system
Photo: İnformatic, CC BY-SA 3.0 (cropped), via Wikimedia Commons
TROUBLESHOOTING

Common problems, causes and fixes

Amplification in no-template controls
Likely causes
  • • Contaminated reagents or water
  • • Amplicon contamination in the lab
  • • Primer-dimers (dye assays)
What to do
  • • Use fresh aliquots and clean the area
  • • Separate pre- and post-PCR areas; never open used plates
  • • Check melt curve; redesign primers or raise annealing temperature
Low efficiency or poor standard curve
Likely causes
  • • Inhibitors in standards or samples
  • • Pipetting error in the dilution series
  • • Degraded standard or wrong annealing temperature
What to do
  • • Dilute the sample or improve extraction clean-up
  • • Use calibrated pipettes and fresh dilutions
  • • Prepare new standard from a qualified stock; optimise annealing
High Cq variation between replicates
Likely causes
  • • Pipetting variation at low volumes
  • • Bubbles or poor sealing
  • • Very low copy number (sampling statistics)
What to do
  • • Use larger reaction volumes or master mixes
  • • Centrifuge the plate and reseal properly
  • • Accept wider limits at the LOQ as defined in validation
Edge wells give different Cq values
Likely causes
  • • Block temperature non-uniformity
  • • Evaporation from poorly sealed edge wells
What to do
  • • Verify block uniformity and service if out of limits
  • • Check seal and heated-lid pressure
Wrong dye signal or crosstalk between channels
Likely causes
  • • Out-of-date or wrong dye calibration
  • • Dye not selected correctly in the run file
What to do
  • • Repeat the pure-dye calibration
  • • Check dye and quencher settings in the plate set-up
Noisy or flat raw signal in some wells
Likely causes
  • • Contaminated or fluorescent block wells
  • • Ageing lamp or LED
  • • Missing passive reference
What to do
  • • Run a background plate and clean the block
  • • Replace the light source
  • • Use a master mix with the right ROX level for this instrument
In LimsPulse · Calibration Management

Keep every qPCR in control: usage logs, calibration and maintenance schedules, and automatic blocking of overdue instruments.

See how