1. Background
  2. Data Collection Methods
  3. Data Quality Control
  4. Summary

MIQE guidelines excerpt from Bustin et al. (2009). Information importance: E = essential; D = desirable. Cq = threshold cycle (CT); NTC = no template control.

Quality Control: Instrumentation and PCR validation

  1. qPCR equipment should be checked regularly (for more information see Life Technologies, 2012)
    1. With age, the excitation source (e.g., halogen lamp or LED) and the emission detector (e.g., photodiode) may lead to variable excitation strength or emission sensitivity across a plate. A passive reference dye may be used to correct for this.
    2. Calibration curves should be generated as part of a maintenance regimen and prior to using new dyes.
  2. PCR validation
    1. Standard calibration curves should be repeated over time to maintain data quality.
      1. Determine whether the efficiency is within the acceptable range of 90 to 110%.
      2. The R2 value is a measure of replicate reproducibility.
      3. Individual reaction efficiencies should be similar.
    2. Melting curve analysis applies to methods that use a fluorescent dye that remains associated with the amplicon (e.g., SYBR® Green I).
      1. The first derivative of a melting curve (-d(RFU)/dT) provides a better graph for analysis.
      2. All amplicon DNA should melt at the same temperature; primer dimers melt at a lower temperature.
      3. Blanks that show a peak indicate the presence of DNA.