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: Data

  1. Standards contain known quantities of the amplicon(s). Important features include:
    1. High quality, not degraded; avoid thawing and refreezing
    2. Correct dilutions used for the calibration curve
    3. Target DNA compatibility
  2. Report qPCR software used and review:
    1. Baseline minimum fluorescence
    2. Threshold value determination
    3. CT values
  3. Two of three replicates must show positive results.
  4. Blanks (no template controls) and positive controls should yield appropriate results
  5. For relative quantification (i.e., normalization), a rationale for the number and choice of reference gene(s) should be given.