Troubleshooting guide for qPCR

qPCR method is very sensitive and, sometimes, some difficulties with optimization can be seen. Please see our qPCR troubleshooting guide for suggestions and help with your specific problems. If you have further questions, please contact Solis BioDyne Technical Support.

Problem Possible solution(s)
Poor signal or no signal
  • Poor quality of template – check template’s purity and integrity, ensure that your reaction doesn’t contain PCR inhibitors; perform a dilution series of the template to determine whether the effect of the inhibitors can be reduced
  • Incompatible, poorly synthesized or degraded primers and/or probe – use different primers and/or probe
  • Template concentration is too low – perform and analyze your reactions on a serially diluted template, start with a high concentration and make dilution series
  • Error in instrument setup – validate that fluorescence data is collected in the right step
  • Non-optimized thermal cycling conditions – check that you use recommended denaturation, annealing, and extension conditions to ensure efficient amplification
Signal in no template control (NTC)
  • Contamination of reaction components -- to avoid contamination, work in dedicated space, keep pre- and post-amplification areas separate, use personal protective equipment, decontaminate your surfaces and equipment, if possible aliquot your reagents into smaller volumes to prevent contamination of stock solutions
  • Primer-dimers: if primer-dimers appear earlier than 35 cycles, redesign your primers; test dilution series of primer concentrations to test whether primer dimers disappear if you use lower/higher primer concentrations
  • Use post-amplification melt curve analysis and agarose gel electrophoresis to confirm presence of non-specific amplification products
Late amplification, high Ct value
  • Non-optimized thermal cycling conditions – check that you use recommended initial activation, denaturation, annealing, and extension conditions to ensure efficient amplification
  • Template concentration is too low – perform and analyze your reactions on a serially diluted template, start with a high concentration and make dilution series
  • Template is degraded – check template’s purity and integrity, re-isolate template from sample material, use freshly prepared template dilution
  • Suboptimal primer design – redesign your primers, use primer-design software, such as Primer3 (bioinfo.ut.ee/primer3) or NCBI Primer-BLAST (ncbi.nlm.nih.gov/tools/primer-blast) to design optimal and target-specific primers
  • Primers concentration is too low -- test dilution series of primer concentrations to determine the optimal amount of primers
Variability between replicates
  • Problem in reaction setup – always prepare master mix, vortex thoroughly, centrifuge briefly and aliquot equal volumes into PCR wells
  • Air bubbles in individual PCR wells – centrifuge reaction samples/plate before running on a qPCR instrument
PCR efficiency below 90% or above 110%
  • Non-optimized thermal cycling conditions – check that you use recommended initial activation, denaturation, annealing, and extension conditions to ensure efficient amplification
  • Suboptimal primer design – redesign your primers, use primer-design software, such as Primer3 (bioinfo.ut.ee/primer3) or NCBI Primer-BLAST (ncbi.nlm.nih.gov/tools/primer-blast) to design optimal and target-specific primers
  • Primers concentration is not optimal – test dilution series of primer concentrations to determine the optimal amount of primers
  • Poor quality of template – check template’s purity and integrity, ensure that your reaction doesn’t contain PCR inhibitors; perform a dilution series of the template to determine whether the effect of the inhibitors can be reduced