Table 2.

Acceptance criteria and recommendations for miR quantification using SYBR^® Green assay in RT‐qPCR

Parameter	Acceptance criteria	Recommendations/ comments
PCR Efficiency	90%‐110%	Absolute quantification: 100 ± 10% Semi‐quantification (eg, comparative quantification of extracellular miR level): ±10% of the mean of batches to be compared; mean does not necessarily have to be 100%
Slope of standard curve	−3.1 to −3.6	Slope up to −4.1 is acceptable, when it is constant within batches to be compared
Correlation coefficient (R2)	≥ 0.98	Correlation coefficient lower than 1 result in inaccuracy quantification of at least one calibration standard
Linearity	see R 2	Within calibration range of extracellular miR concentrations; Aa [%] of calculation standards ± 30%, but can be higher when R 2 < 1.0
Specificity	Tmb ± 1°C	Melting curve analysis of all batches
Sensitivity	ALLOQ c ≤ 30%	Accuracy of standard curve can be higher when R2 < 1.0
Repeatability	CVd ≤ 25% CVLLOQ ≤ 30%	CV of quantified concentrations, not Cq‐values
Reproducibility	CV ≤ 35% CVLLOQ ≤ 40%	CV of quantified concentrations, not Cq‐values
Trueness	AQC ± 30%	Accuracy of low QCse is not within acceptance criteria

^aAccuracy.

^bMelting temperature.

^cLower limit of quantification.

^dCoefficient of variation.

^eQuality control.