Pfaffl Expression Ratio

Calculate Pfaffl expression ratio with independent primer efficiencies.

Description

Efficiency-corrected qPCR compares control-to-treatment Ct shifts for target and reference assays.

Efficiency-corrected qPCR compares control-to-treatment Ct shifts for target and reference assays. Each efficiency is an amplification factor per cycle, such as 2 for ideal doubling, rather than a percentage.^{[1, 2]}

Calculate Pfaffl expression ratio with independent primer efficiencies.

When to use Pfaffl Expression Ratio

Use this tool to calculate Pfaffl expression ratio with independent primer efficiencies.

Prepare the upstream counts, expression values, sequences or record lists requested by this tool. Keep corresponding features in the same order and confirm count units, transformations and caller-selected criteria.

How to use Pfaffl Expression Ratio and understand its results

Prepare the upstream counts, expression values, sequences or record lists requested by this tool. Keep corresponding features in the same order and confirm count units, transformations and caller-selected criteria.
Enter targetEfficiency, referenceEfficiency, targetControlCt, targetTreatmentCt, referenceControlCt, referenceTreatmentCt using the field table and calculation rules, then run the tool.
Read targetFold, referenceFold, expressionRatio. The explanation below defines the units, signs, ordering and any flags or placeholder values.
If the tool reports an input issue, correct the values and run it again. Apply the stated assumptions when interpreting a successfully returned result.

Input field	Meaning	Value type; see rules below	Requirement
targetEfficiency	Target efficiency	number	Required
referenceEfficiency	Reference efficiency	number	Required
targetControlCt	Target control ct	number	Required
targetTreatmentCt	Target treatment ct	number	Required
referenceControlCt	Reference control ct	number	Required
referenceTreatmentCt	Reference treatment ct	number	Required

Enter positive target/reference amplification factors and the four finite Ct values using the same condition convention. Compute each assay's fold factor and divide target by reference. Return both fold factors and the expression ratio.

ratio=targetEfficiency^(targetControlCt−targetTreatmentCt)/referenceEfficiency^(referenceControlCt−referenceTreatmentCt)

Units, indexing, boundary conditions and exceptional cases are defined in the calculation rules above.

Output field	Meaning	Value type
targetFold	Target fold	number
referenceFold	Reference fold	number
expressionRatio	Expression ratio	number

json

{
  "input": {
    "targetEfficiency": 2,
    "referenceEfficiency": 2,
    "targetControlCt": 25,
    "targetTreatmentCt": 23,
    "referenceControlCt": 20,
    "referenceTreatmentCt": 20
  },
  "output": {
    "targetFold": 4,
    "referenceFold": 1,
    "expressionRatio": 4
  }
}

Limitations and assumptions

The efficiencies are supplied and not estimated or restricted to a typical experimental range. No replicate uncertainty, reference stability, undetected-Ct handling or assay validation is included.

Numeric inputs must be finite. Follow the calculation-specific rules for overflow, underflow and exceptional cases. Missing required fields, extra fields, wrong value types and violations of the calculation rules are input errors.

Alternative or Complementary methods

Use Global Mean Delta CT: Normalize target Ct values to a reference-gene global mean. For a complementary task, use Bestkeeper Stability: Screen reference-gene Ct stability by replicate standard deviation.

Further background: Efficiency-corrected relative quantification in real-time RT-PCR.

References

Pfaffl Expression Ratio — tool.io
Efficiency-corrected relative quantification in real-time RT-PCR — Pfaffl, Nucleic Acids Research

Similar or alternative tools

Global Mean Delta CT
Normalize target Ct values to a reference-gene global mean.
Bestkeeper Stability
Screen reference-gene Ct stability by replicate standard deviation.