Luna® Universal qPCR & RT-qPCR
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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.

Videos

  1. Luna Dots In Boxes

    Learn how we analyzed plates and plates of qPCR data, and how Luna stacks up to the competition.

  2. overview of qPCR

    Overview of qPCR

    Learn the basics of qPCR in this short animation.                                                                                                  

  3. NEBTv_Ep16_Thumb

    NEB® TV Ep. 16 – Overview and Applications of qPCR

    Learn the basics of qPCR, and hear more about our Luna products, including our novel method for data analysis.

Benefit from rigorous testing

  • All Luna reagents were evaluated for specificity, sensitivity, efficiency, accuracy, linearity and reproducibility, and optimized for performance
  • Development of novel "dots in boxes" analysis method for concise visual presentation of comprehensive evaluations

Use with ALL your targets

  • Extensive panel testing and evaluation ensures reproducible results
  • Luna reagents are optimized for strong performance across a wide range of targets and templates, including those of varied length, GC-content and abundance

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:

  1. Linearity of amplification, as indicated by the R2 standard curve
  2. Reproducibility, as indicated by the consistency of triplicate Cq values for each input concentration
  3. Fluorescence consistency, as indicated by similar endpoint fluorescence (RFUmax)
  4. Curve steepness
  5. 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.

Breaking it down: how we translate qPCR data into "dots in boxes"

dots in boxes

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

Luna Evaluation of commercially available qPCR

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.


A set of 24 targets demonstrates the most sensitive quantitation from the Luna Cell Ready One-Step RT-qPCR Kit compared to the commercially available cell lysis One-Step RT-qPCR kits.



Approximately 2,500 A549 cells were lysed in 50 µl Luna Cell Ready lysis reactions (NEB #E3032) using standard reaction conditions or with commercially available kits from Bio-Rad (SingleShot SYBR Green One-Step Kit, #172-5095), Qiagen (FastLane Cell SYBR Green Kit, #216213) and Thermo Fisher (Cells-to-CT 1-Step PowerSYBR Green Kit, A25600) following manufacturer-recommended protocols. Two biological replicates were processed for each kit. 24 genes of interest were then quantitated using the One-Step RT-qPCR module from each kit with 1 μl of cell lysate as input (equal to 50 cells in a 20 µl RT-qPCR reaction), with duplicate reactions for each biological sample. Average Cqs are shown for NEB (closed orange circles), BioRad (open squares), Qiagen (open triangles) and Thermo Fisher (crosses). To standardize results, 12% of total fluorescence was set as a threshold. The Luna Cell Ready One-Step RT-qPCR kit shows the earliest Cq for 23/24 genes across variable expression levels, with an average of 2.3 Cq faster than Bio-Rad, 3.8 Cq faster than Qiagen and 3.6 Cq faster than Thermo Fisher. 


Available products:

Luna Universal qPCR Master Mix

Rapid, sensitive, and precise dye-based qPCR detection and quantitation of target DNA and cDNA sequences 
Luna Universal Probe qPCR Master Mix

Rapid, sensitive, and precise probe-based qPCR detection and quantitation of target DNA and cDNA sequences
Luna Universal One-Step RT-qPCR Kit

Includes everything you need for rapid, sensitive, and precise dye-based qPCR detection and quantitation of RNA targets
Luna Universal Probe One-Step RT-qPCR Kit

Includes everything you need for rapid, sensitive, and precise probe-based qPCR detection and quantitation of RNA targets
Luna Probe One-Step RT-qPCR 4X Mix with UDG

4X mix enables sensitive detection of target RNA sequences, with robust performance in multiplex applications of up to 5 targets
LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG

Lyophilized format enables sensitive detection of target RNA sequences with room temperature stability
Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit

For real-time qualitative detection of SARS-CoV-2 nucleic acid using hydrolysis probes
LunaScript RT SuperMix Kit

Single tube with blue tracking dye includes everything needed for first strand cDNA synthesis in two-step RT-qPCR workflows
LunaScript RT Master Mix Kit (Primer-free)

Single-tube 5X master mix contains Luna Reverse Transcriptase, dNTPs, and Murine RNase Inhibitor
Luna Cell Ready One-Step RT-qPCR Kit

All the necessary components for direct, dye-based RNA detection and quantitation from cell lysate, bypassing the need for RNA extraction and purification
Luna Cell Ready Probe One-Step RT-qPCR Kit

All the necessary components for direct, probe-based RNA detection and quantitation from cell lysate, bypassing the need for RNA extraction and purification
Luna Cell Ready Lysis Module

Coordinates cell lysis, RNA release, and genomic DNA removal simultaneously, resulting in cell lysate ready for direct RT-qPCR analysis with Luna Universal One-Step RT-qPCR kits

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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.
QIAGEN® is a registered trademark of Qiagen, Inc.
BIO-RAD® is a registered trademark of Bio-Rad Laboratories, Inc.