AAT Bioquest

How do I design primers for qPCR?

Posted May 12, 2023


Primer design is a crucial aspect of qPCR for reliable gene expression analysis.  Poorly annealing primers or primers that bind to multiple sequences during amplification can adversely affect the quality and accuracy of qPCR results.

Here are some guidelines to follow when designing primers for your qPCR assay:

  • Aim for primers with a GC content between 50% and 60%. 
  • Place Gs and Cs at the ends of the primers.
  • Avoid repeat sequences of Gs or Cs that are longer than three bases.
  • Use the nearest-neighbor method to achieve a melting temperature (Tm) between 50°C and 65°C. Online Tm calculators using a salt concentration of 50 mM and oligonucleotide concentration of 300 nM can be helpful.
  • Ensure no 3' complementarity between the sequences of forward and reverse primers to prevent primer-dimer formation.
  • Avoid secondary structure by placing primers outside secondary structure in target sequence regions if necessary.
  • Verify primer specificity using tools such as the Basic Local Alignment Search Tool (BLAST).
Additional resources

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Real-Time PCR (qPCR)

ROX Reference Dye *50X fluorescence reference solution for PCR reactions*