Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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

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 

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Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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库存
货号 浓度 规格 目录价 北京 上海 广州 成都 苏州
M3019S 4 X 200 reactions ¥3,719.00 有 无 无 无 无
M3019L 4 X 500 reactions ¥8,379.00 无 无 无 无 无
M3019X 4 X 1,000 reactions ¥14,889.00 无 无 无 无 无
M3019E 4 X 2,000 reactions ¥26,279.00 无 无 无 无 无
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Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs Want to evaluate this product? Contact us for a sample Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs Need a custom/large volume order? Contact us

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产品信息

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

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.

Luna® Probe One-Step RT-qPCR 4X Mix with UDG | NEB酶试剂 New England Biolabs

产品类别:
Luna® qPCR & RT-qPCR Products

应用:
qPCR & RT-qPCR,
RT-qPCR, RT-PCR and cDNA Synthesis

  • 产品组分信息

    产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • M3019S     -20    
        Luna® Probe One-Step RT-qPCR 4X Mix with UDG M3019SVIAL -20 2 x 0.53 ml 4 X
        Nuclease-free Water B1502AVIAL -20 2 x 1.5 ml 不适用
    • M3019L     -20    
        Luna® Probe One-Step RT-qPCR 4X Mix with UDG M3019LVIAL -20 2 x 1.25 ml 4 X
        Nuclease-free Water B1502AVIAL -20 4 x 1.5 ml 不适用
    • M3019X     -20    
        Luna® Probe One-Step RT-qPCR 4X Mix with UDG M3019LVIAL -20 4 x 1.25 ml 4 X
        Nuclease-free Water B1502AVIAL -20 8 x 1.5 ml 不适用
    • M3019E     -20    
        Luna® Probe One-Step RT-qPCR 4X Mix with UDG M3019EVIAL -20 1 x 10.5 ml 4 X
        Nuclease-free Water B1502EVIAL -20 1 x 25 ml 不适用

  • 特性和用法

  • 相关产品

    相关产品

    • LunaScript™ RT SuperMix Kit
    • Luna® Probe One-Step RT-qPCR 4X Mix with UDG (No ROX)
    • Luna® Universal Probe One-Step RT-qPCR Kit
    • Luna® Probe One-Step RT-qPCR Kit (No ROX)
    • Monarch® Total RNA Miniprep Kit

操作说明、说明书 & 用法

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

  • 应用实例
    • The Luna 4X RT-qPCR Mix and Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit enable high throughput, automated detection workflows
    • Optimized conditions for the CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay using Luna One-Step RT-qPCR Reagents

FAQs & 问题解决指南

  • 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 cycling conditions 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 Mix that contain a 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 RNA Cleanup Kits (NEB # T2030, #T2040, #T2050) compatible with Luna RT-qPCR reagents?
    21. What are the differences between the Luna One-Step RT-qPCR products (NEB #E3006, #E3007 and #M3019)?
    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 One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) product for viral detection?

引用 & 技术文献

  • 专题文章
    • Using aptamers to control enzyme activities: Hot Start Taq and beyond

质控、安全 & 法规

  • 质控分析
    每一新批次的 NEB 产品都要经过质控，以满足指定的质量标准，对特定产品的质量标准和每一批次的检测数据可以通过产品说明表格、COA、产品信息卡或者产品手册进行查阅或下载。关于 NEB 产品质控的详细信息，可从此处查阅。

  • 产品说明与变更通知

    产品说明与变更通知

    产品说明表包含产品的储存温度、有效期和规格，这些文件的命名规则如下：[货号]_[规格]_[版本]

    • M3019E_v1
    • M3019S_L_X_v1

  • CoA
    CoA 文件包含单个批次产品的储存温度、有效期和质控，这些文件的命名规则如下： [货号]_[规格]_[版本]_[Lot#]

    • M3019G_v0_10083028
    • M3019E_v1_10086188
    • M3019S_v1_10083018
    • M3019X_v1_10083022
    • M3019S_v1_10105787
    • M3019E_v1_10105176
    • M3019L_v1_10107199
    • M3019G_v0_10107203
    • M3019X_v1_10107200
    • M3019S_v1_10108249
    • M3019E_v1_10108244
    • M3019L_v1_10109897
    • M3019E_v1_10111866
    • M3019L_v1_10111050
    • M3019L_v1_10116524
    • M3019X_v1_10117878
    • M3019L_v1_10118240
    • M3019G_v0_10119933
    • M3019L_v1_10120893
    • M3019L_v1_10121214
    • M3019X_v1_10118241
    • M3019E_v1_10120298
    • M3019E_v1_10122640
    • M3019S_v1_10123462
    • M3019G_v0_10124445
    • M3019X_v1_10123082
    • M3019E_v1_10123103
    • M3019X_v1_10125213
    • M3019L_v1_10123083
    • M3019G_v0_10129812
    • M3019L_v1_10125064
    • M3019X_v1_10131110
    • M3019E_v1_10083030
    • M3019X_v1_10132781
    • M3019L_v1_10131847
    • M3019S_v1_10129860
    • M3019X_v1_10135526
    • M3019G_v0_10136231
    • M3019L_v1_10135836
    • M3019L_v1_10137587
    • M3019L_v1_10083020
    • M3019E_v1_10131850
    • M3019L_v1_10138414
    • M3019E_v1_10140705
    • M3019S_v1_10140630
    • M3019L_v1_10140413
    • M3019X_v1_10138429
    • M3019G_v0_10142511
    • M3019G_v0_10143056
    • M3019X_v1_10145816
    • M3019L_v1_10145818
    • M3019E_v1_10146690
    • M3019G_v0_10149553
    • M3019G_v0_10157112
    • M3019E_v1_10156887
    • M3019L_v1_10157020
    • M3019X_v1_10159765
    • M3019G_v1_10162776

  • SDS
    以下 SDS 文件可以帮助您安全地使用该产品

    • Luna® Probe One-Step RT-qPCR 4X Mix with UDG
    • Nuclease-free Water