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Monday, June 24, 2013

MIQE Guidelines for representing and analysing qPCR data

MIQE guidelines are a set of guidelines for the analysis and representation of qPCR or Real Time PCRdata, inaccurate data and statistical analysis can be misleading and one need to follow the MIQE guidelines for representing and analysing qPCR data

Standard terms to be used for Real Time PCR data publications:

  • qPCR for quantitative PCR and RT – PCR for reverse transcription PCR. The term RT-PCR should not be used to represent real time PCR.

  • genes used for normalization should be referred to as reference genes not house keeping genes.

  • TaqMan probes should be referred to as hydrolysis probe.

  • The term FRET probe (fluorescence resonance energy transfer probe) refers to a generic mechanism in which emission/quenching relies on the interaction between the electron-excitation states of 2 fluorescent dye molecules. LightCycler-type probes should be referred to as dual hybridization probes.

  • Quantification is the correct term to use not quantitation.

  • According to RDML (Real-Time PCR Data Markup Language) data standard quantification cycle (Cq) is the correct term to use, threshold Cycle Ct, Crossing point (Cp) and take-off point(TOP) even though all these are coined by manufactures of PCR machines for differentiation.

Analytical sensitivity refers to the minimum number of copies in a sample that can be measured accurately with an assay, whereas clinical sensitivity is the percentage of individuals with a given disorder whom the assay identifies as positive for that condition. Typically, sensitivity is expressed as the limit of detection (LOD), which is the concentration that can be detected with reasonable certainty with a given analytical procedure. The most sensitive LOD theoretically possible is 3 copies per PCR.

Analytical specificity refers to the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample. Diagnostic specificity is the percentage of individuals without a given condition whom the assay identifies as negative for that condition.

Accuracy refers to the difference between experimentally measured and actual concentrations, presented as fold changes or copy number estimates.

Repeatability (short-term precision or intraassay variance) refers to the precision and robustness of the assay with the same samples repeatedly analyzed in the same assay. It may be expressed as the SD for the Cq variance. Alternatively, the SD or the CV for copy number or concentration variance may be used. CVs should not be used with Cqs.

Reproducibility (long-term precision or interassay variance) refers to the variation in results between runs or between different laboratories and is typically expressed as the SD or CV of copy numbers or concentrations. Cq values generated from different runs are subject to inherent interrun variation hence, reporting interrun Cq variation is not appropriate.


MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Special Report