All NEB products undergo rigorous testing to ensure optimal performance, and Luna is no exception. We took into consideration numerous important traits when evaluating qPCR, including specificity, sensitivity, accuracy and reproducibility, to develop best-in-class qPCR products. Furthermore, we did a comprehensive evaluation of many commercially-available qPCR and RT-qPCR products, and developed a method of analysis that allows you to quickly compare and evaluate the performance of these products. We wanted to be sure that Luna products will perform to your expectations for all of your targets.
Evaluating qPCR results: capturing performance as "dots in boxes"
NEB has developed a method to better evaluate the large amount of qPCR data generated in an experiment. The output of this analysis is known as a dot plot, and captures the key features of a successful, high-quality qPCR experiment as a single point. This method of analysis allows many targets and conditions to be compared in a single graph.
For each experiment, triplicate reactions are set up across a five-log range of input template concentrations (Amplification plot, bottom-left). Three non-template control (NTC) reactions are also included, for a total of 18 reactions per condition/target. Efficiency (%) is calculated (Standard curve, top-left) and is plotted against ΔCq (dot plot, center), which is the difference between the average Cq of the NTC and the lowest input. This parameter captures both detection of the lowest input and non-template amplification. Acceptable performance criteria are defined as an Efficiency of 90-110% and a ΔCq of ≥ 3 (green box).
Other performance criteria are captured using a 5-point Quality Score (top-right). Included are:
- Linearity of amplification, as indicated by the R2 standard curve
- Reproducibility, as indicated by the consistency of triplicate Cq values for each input concentration
- Fluorescence consistency, as indicated by similar endpoint fluorescence (RFUmax)
- Curve steepness
- Sigmoid curve shape
Quality Score is represented by the size and fill of the plotted dot, with experiments that pass all performance criteria represented by a solid dot within the box. These scoring methods were built upon the MIQE qPCR/RT-qPCR guidelines (Bustin, S.A. et al. (2009) Clin Chem. 55, 611-22 and Trombley Hall, A. et al. (2013) PLoS One 8(9):e73845). An example of this analysis is shown below.
See how Luna products stack up
Comprehensive market-wide evaluation of qPCR and RT-qPCR products reveals that Luna outperforms the competition.
Evaluation of commercially-available qPCR products demonstrates robustness and specificity of Luna
Panels of qPCR/RT-qPCR targets varying in abundance, length, and %GC were used to evaluate the performance of a variety of commercial reagents. The bar graph summarizes % of targets that met acceptable performance criteria of 90 – 110% Efficiency, ΔCq ≥ 3 and Quality Score > 3 (indicated by green box on dot plot and line on Quality Score key) from individual experiments, evaluated as described above. Each target panel was run by 2 users and according to manufacturer's protocols.
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APPLIED BIOSYSTEMS™ is a trademark of Thermo Fisher Scientific, Inc., STEPONEPLUS® and QUANTSTUDIO® are registered trademarks of Thermo Fisher Scientific, Inc.
CFX96 TOUCH™ is a trademark of Bio-Rad Laboratories, Inc.
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BIO-RAD® is a registered trademark of Bio-Rad Laboratories, Inc.