Product Class: Other

Luna® Probe One-Step RT-qPCR 4X Mix with UDG


Catalog #M3019

Product Introduction

The Luna Probe One-Step RT-qPCR 4X Mix with UDG enables sensitive detection of target RNA sequences with robust performance in multiplex applications of up to 5 targets. This product is also offered in a No ROX formulation for instruments that do not require the ROX passive reference dye and in a lyophilized format for streamlined assay development.

  • Simplify your reaction setup with a single-tube master mix format 
  • Increase sensitivity of your RT-qPCR, as 4X concentration allows for more sample input
  • Maximize your throughput by multiplexing up to 5 targets
  • Luna WarmStart® RT paired with Hot Start Taq increases reaction specificity and robustness, enabling room temperature setup
  • Reduce risk of carryover contamination with thermolabile UDG and dUTP included in the mix
  • Eliminate pipetting errors with non-interfering, visible blue tracking dye 
  • Learn how NEB is supporting COVID-19 research with a variety of RT-qPCR virus detection options 

Product Information

Description


The Luna Probe One-Step RT-qPCR 4X Mix with UDG is designed for real-time detection of target RNA sequences using hydrolysis probes. In a single tube, RNA is first converted to cDNA by a reverse transcriptase, then a DNA-dependent DNA polymerase amplifies the cDNA, enabling quantitation via real time or quantitative PCR (qPCR). Probe-based qPCR/RT-qPCR monitors an increase in fluorescence upon 5′ to 3′ exonuclease cleavage of a quenched, target-specific probe to measure DNA amplification at each cycle of a PCR. At a point where the fluorescence signal is confidently detected over the background fluorescence, a quantification cycle or Cq value can be determined. Cq values can be used to evaluate relative target abundance between two or more samples.

Due to the one-step RT-qPCR workflow and probe-based detection chemistry, a comparison can be made to the Luna Probe One-Step RT-qPCR Kit (NEB #E3006).

Figure 1: A new product choice for one-step RT-qPCR assays





The Luna Probe One-Step RT-qPCR Mix with UDG is supplied at a 4X concentration and enables higher amounts of sample input, which is relevant for applications where RNA present in low abundance is of interest, such as pathogen detection. Performance in multiplexing applications has been optimized, with sensitive, linear detection achieved for up to 5 targets across a range of inputs. The mix consolidates the necessary components for one-step RT-qPCR into a single tube, including Luna WarmStart RT, Hot Start Taq DNA Polymerase, dNTPs, and Murine RNase Inhibitor in an optimized buffer. Combining Hot Start Taq DNA Polymerase with a novel, WarmStart-activated reverse transcriptase allows for dual control of enzyme activity via reversible, aptamer-based inhibition. This temperature-dependent activation helps to prevent undesirable non-specific priming and extension prior to thermocycling, providing added security for setting up reactions at room temperature. The engineered Luna WarmStart RT also possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C. Additionally, the mix is formulated with a unique passive reference dye that is compatible across a variety of instrument platforms, including those that require a high or low ROX reference signal. 

Figure 2: Robust amplification and detection of different viral RNA with Luna Probe One-Step RT-qPCR 4X Mix with UDG



RT-qPCR targeting SARS-CoV-2 (N1 target) and H. influenza H1N1 (HA target) was performed using the Luna Probe One-Step RT-qPCR Mix with UDG. Performance in 20 μl reactions was evaluated in two real-time instruments over a 5-log range of (A) 10–100,000 copies Synthetic SARS-CoV-2 RNA Control 2 (Twist Biosciences, SKU: 102024) diluted in 10 ng of Jurkat total RNA (BioChain, #R1255815-50) and (B) 12.4–124,000 copies Influenza A (H1N1) RNA (ATCC®VR-95DQ™) diluted in 10 ng Jurkat total RNA. Sensitive, linear performance can be observed in the amplification of both viral targets.




Figure 3: Multiplex detection (5 targets) with the Luna Probe One-Step RT-qPCR 4X Mix with UDG



Multiplex RT-qPCR was performed using the Luna Probe One-Step RT-qPCR 4X Mix with UDG over a 5-log range of Jurkat total RNA (100 ng to 10 pg) on a Bio-Rad CFX96 real-time instrument. Amplification standard curves and efficiencies for each of the 5 human targets are shown. Reactions (20 μl) included primers and probes at 200 nM each, and followed the product recommended cycling conditions. All five targets were detected linearly in the multiplex reactions with strong efficiency and R2 values. 




Figure 4: The Luna Probe One-Step RT-qPCR 4X Mix with UDG outperforms comparable products



One-step RT-qPCR was tested on 8 RT-qPCR targets (indicated by color) varying in abundance, length, and %GC. Data was collected on multiple days by two users according to manufacturer’s recommendations using the Applied Biosystems™ QuantStudio™ 6 real-time PCR system. Results were evaluated for efficiency, low input detection and lack of non-template amplification (where ΔCq = average Cq of non-template control – average Cq of lowest input). In addition, consistency, reproducibility and overall curve quality were assessed based on metrics described previously (Quality Score). Although both products performed reasonably well, NEB’s Luna Probe RT-qPCR 4X Mix with UDG outperformed the TaqPath 1-Step RT-qPCR Master Mix, CG, as evidenced by the higher number of experiments whose results fell in the green box.




The sensitivity of RT-qPCR makes it important to minimize DNA contamination wherever possible. The inclusion of dUTP and thermolabile UDG prevents carryover contamination, where unintended product of a previous amplification can serve as the substrate of a subsequent reaction. The thermolabile UDG is completely inactivated at temperatures above 50°C, thereby having no effect on qPCR performance.

Figure 5: Evaluation of RT-qPCR carryover prevention



To evaluate the capacity of carryover product digestion, a uracil-containing RT-qPCR product was generated using the Luna One-Step Probe RT-qPCR kit. Approximately 105 copies of the uracil-containing product were used as template for subsequent qPCR reactions using three different RT-qPCR master mixes (all containing UDG). Following manufacturer’s recommended protocols, incubation at 25˚C at the start of the 2nd reaction enables UDG to degrade the dU-containing input, reducing its ability to contribute to a (false) positive signal. The ΔCq value is the cycle difference between carryover treatment and no carryover treatment of the same input. Larger ΔCq values indicate more efficient carryover product digestion. After decontamination using the Luna mix, full product digestion was observed in one sample and a large ΔCq was observed in the second. Note that the amount of DNA present in true contamination events is difficult to assess/predict.




A non-fluorescent visible blue tracking dye is included to assist in pipetting into clear vessels. The tracking dye does not overlap spectrally with fluorophores commonly used in qPCR and does not interfere with real-time detection.

 

The Luna Probe One-Step RT-qPCR Master Mix with UDG contains an inert blue tracking dye to eliminate pipetting errors.





This product is related to the following categories:
Luna® qPCR & RT-qPCR
This product can be used in the following applications:
qPCR & RT-qPCR

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Protocol for Luna® Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019)

Application Notes

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. How do I use qPCR to determine the concentration of my material?
  2. Can I set up my Luna® RT-qPCR at room temperature?
  3. Should I use probe- or dye-based detection for my qPCR assays?
  4. How should I design primers for Luna® qPCR?
  5. How long should my amplicon be for qPCR?
  6. Why is the Luna® qPCR Mix blue? Will this dye interfere with detection?
  7. Can I run the Luna® qPCR Mix on my qPCR instrument?
  8. Can I use fast instrument settings with the Luna® qPCR Mix?
  9. Do I need to add ROX?
  10. What RNA samples can be used in RT-qPCR with the Luna® Mix?
  11. How much primer and probe should I use with the Luna® Universal Probe qPCR Master Mix?
  12. Can I use shorter cycling times?
  13. How much RNA template should I use in my RT-qPCR reaction?
  14. Can I run multiplex reactions with the Luna® Probe One-Step RT-qPCR Kits? Do I need to change my reaction conditions?
  15. Can I use a ROX-labeled probe with the Luna® Probe Mixes that contain a universal ROX reference dye?
  16. Can alternative probe based detection strategies be used with the Luna® Probe Mix?
  17. What temperature should I use for cDNA synthesis with Luna® RT-qPCR kits?
  18. Can I use longer targets in one-step RT-qPCR?
  19. Is the Monarch® Total RNA Miniprep Kit compatible with Luna® RT-qPCR Reagents?
  20. Are the Monarch Spin RNA Cleanup Kits compatible with Luna RT-qPCR reagents?
  21. What are the differences between the Luna One-Step RT-qPCR products (NEB #E3006, #E3007, #M3019, #M3029 and #L4001)?
  22. Other companies recommend a 2-min incubation at 25°C for carryover prevention, is a 30-sec incubation at 25°C sufficient?
  23. How can I perform a no-RT Control? 
  24. Can I use the Luna Probe RT-qPCR mixes for viral detection?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

Nucleic acid-based aptamers for use with thermophilic DNA polymerases and reverse transcriptases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the Luna® Probe One-Step RT-qPCR 4X Mix with UDG product for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.

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