产品信息

Luna 探针一步法 RT-qPCR 试剂盒（无 ROX）经过优化，适用于采用水解探针法的靶标 RNA 序列实时定量。一步法 RT-qPCR 为 RNA 检测和定量提供了一种便捷、强大的方法。在同一管中，RNA 首先由反转录酶转化为 cDNA，然后使用 DNA 依赖型 DNA 聚合酶扩增 cDNA，从而可以进行 qPCR 定量。探针法 qPCR/RT-qPCR 原理是利用聚合酶的 5´→3´ 核酸外切酶活性，将淬灭的目标特异性探针切断发出荧光，并实时监测荧光值的增加，以测量 PCR 每个循环中的 DNA 扩增。通过荧光信号显著超过背景荧光所经历的循环数，可以确定 C_q 值。C_q 值可用于评估两个或多个样品之间的靶基因相对丰度，或者参考通过已知浓度样品绘制的标准曲线，对未知样品绝对定量。

在 Luna 探针一步法 RT-qPCR 试剂盒（无 ROX）中，联合使用 Taq 热启动 DNA 聚合酶与新型 WarmStart 反转录酶，通过适配体可逆抑制来双重控制酶活性。这种温控活化机制可避免热循环前的非特异扩增，从而让在室温下建立体系更安全。经改造的 Luna WarmStart 反转录酶的热稳定性比许多反转录酶更高，其最佳反应温度为 55℃。对于困难靶标/模板，最多可将反转录步骤的温度升至 60℃，且不会影响 Luna 性能。
 
注意：为确保 Luna WarmStart 反转录酶完全激活，建议温育温度不低于 50℃。

Luna 探针一步法 RT-qPCR 试剂盒（无 ROX）浓度为 2X，包含 Taq 热启动 DNA 聚合酶、dNTP 和所有必需的缓冲液成分。配方中不含参比染料，与不需要 ROX 的仪器兼容（如果需要 ROX 校正，可自行添加 ROX 染料）。反应预混液包含可防止交叉污染的 dUTP，以及有助于观察反应体系建立的非荧光可见示踪染料。该示踪染料与 qPCR 常用荧光团的光谱没有重叠，因此不会干扰实时检测。

Luna WarmStart 反转录酶预混液的浓度为 20X，其中包含 Luna WarmStart 反转录酶以及用于防止 RNA 降解的小鼠 RNase 抑制剂（详情请见产品手册中的模板制备）。适用于各种 RNA 样品类型（总 RNA、poly（A）-RNA 等）和来源。

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图 1：NEB 的 Luna 通用探针一步法 RT-qPCR 试剂盒*具有更高的灵敏度、更出众的可重复性及更优异的 RT-qPCR 表现

图 2：NEB 的 Luna 通用探针一步法 RT-qPCR 试剂盒*在多重检测中展现强大功效

图 3：通过与市售的探针法 RT-qPCR 试剂相比，Luna 产品展现出更好的稳定性和特异性

*数据来源于 Luna 通用探针一步法 RT-qPCR 试剂盒（NEB #E3006）。Luna 探针一步法 RT-qPCR 试剂盒（无 ROX）（NEB #E3007）的性能与之相同。

产品类别:

Luna® qPCR & RT-qPCR Products,

PCR, qPCR & Amplification Technologies Products

应用:

qPCR & RT-qPCR,

PCR,

DNA Amplification, PCR & qPCR

• 试剂盒组成

  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
  E3007E     -20       Luna® WarmStart® RT Enzyme Mix M3002EVIAL -20 2 x 1.25 ml 20 X   Luna® Probe One-Step Reaction Mix (No ROX) M3007EVIAL -20 1 x 25 ml 2 X   Nuclease-free Water B1502EVIAL -20 1 x 25 ml Not Applicable

• 相关产品

  相关产品

  • 南极热敏 UDG
  • Luna^® 通用一步法 RT-qPCR 试剂盒
  • Luna^® 通用 qPCR 预混液
  • Luna^® 通用探针法 qPCR 预混液

• 注意事项

  1. Primer Design
     The use of qPCR primer design software (e.g., Primer3) maximizes the likelihood of amplification success while minimizing nonspecific amplification and primer dimers. Targets with balanced GC/AT content (40–60%) tend to amplify most efficiently. Where possible, enter sufficient sequence around the area of interest to permit robust primer design and use search criteria that permit cross-reference against relevant sequence databases (to avoid potential off-target amplification). It is advisable to design primers across known RNA splicing sites in order to prevent amplification from genomic DNA.
  2. Primer and Probe Concentrations
     For most targets, a final concentration of 400 nM (each primer) will provide optimum performance. If needed, primer concentrations can be optimized between 100–900 nM. Probe should be included at 200 nM for best results. Probe concentration can be optimized in the range of 100–500 nM.
  3. Multiplexing
     When determining which fluorophores to include in a multiplex reaction, be sure to choose compatible reporter dyes and quenchers (e.g., those that can be accommodated by the chosen real-time instrument with minimal overlap in fluorescence spectra). For ROX-dependent instruments, avoid ROX-labeled probes. Include 400 nM of forward and reverse primers and 200 nM probe for each target to be detected in the reaction. For targets that differ significantly in abundance, use of a lower primer concentration (e.g. 200 nM) for the more abundant target(s) is recommended. Adjust concentrations if necessary based on performance (primer 100–900 nM, probe 100–500 nM). When loading a qPCR protocol onto the real-time instrument, be sure to select the appropriate optical channels, as some instruments have a single channel recording mode that would prevent multiplex data collection and analysis. The functionality of the primer and probe sets should be tested individually before attempting a multiplex reaction.
  4. Amplicon Length
     To ensure successful and consistent qPCR results, it is important to maximize PCR efficiency. An important aspect of this is the design of short PCR amplicons (typically 70–200 bp). Some optimization may be required for targets that exceed that range.
  5. Template Preparation and Concentration
     Luna RT-qPCR is compatible with RNA samples prepared through typical nucleic acid purification methods. Prepared RNA should be stored in an EDTA-containing buffer (e.g., 1X TE) for long-term stability, and dilutions should be freshly prepared for a qPCR experiment in either TE or water. Note that the quality of RNA templates can greatly affect RT-qPCR efficiency. RNA should be handled with appropriate precautions to prevent RNase or DNase contamination. Use of nuclease-free water (provided) is strongly recommended. Where useful, RNA may be treated with DNase I to remove contaminating genomic DNA.
     Generally, a useful concentration of standard and unknown material will be in the range of 10⁸ copies to 10 copies. Note that for dilutions in the single-copy range, some samples will contain multiple copies and some will have none, as defined by the Poisson distribution. For total RNA, Luna One-Step Kits can provide linear quantitation over an 8-order input range of 1 μg – 0.1 pg. For most targets, a standard input range of 100 ng – 10 pg total RNA is recommended. For purified mRNA, input of ≤ 100 ng is recommended. For in vitro-transcribed RNA, input of ≤ 10⁹ copies is recommended.
  6. ROX Reference Dye

     Some real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by machine limitations such as “edge effect”, bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. However, ROX normalization does little to the variations caused by pipetting errors of templates/primers, heterogeneous mixing, and evaporation/condensation issues.

     A universal passive reference dye is included in the following Luna^® qPCR products: Luna Universal qPCR Master Mix (NEB #M3003), Luna Universal Probe qPCR Master Mix (NEB #M3004), Luna Universal One-Step RT-qPCR Kit (NEB #E3005), and Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006). These products support broad instrument compatibility (High-ROX, Low-ROX, ROX-independent) so no additional ROX is required for normalization.

     The Luna Probe One-Step RT-qPCR Kit (No ROX) (E3007) contains no reference dye and is compatible with any instrument that does not require ROX. If ROX normalization is needed, ROX can be added. Please refer to instrument manufacturer’s instructions for greater details.

  7. Carryover Contamination Prevention
     RT-qPCR is an extremely sensitive method, and contamination in new RT-qPCR assays with products from previous amplification reactions can cause a variety of issues, such as false positive results and a decrease in sensitivity. The best way to prevent this “carryover” contamination is to practice good laboratory procedures and avoid opening the reaction vessel post amplification. However, to accommodate situations where additional anti-contamination measures are desired, Luna qPCR mixes contains a mixture of dUTP/dTTP that results in the incorporation of dU into the DNA product during amplification. Pretreatment of qPCR/RT-qPCR experiments with uracil DNA glycosylase (UDG) will eliminate previously-amplified uracil-containing products by excising the uracil base to produce a non-amplifiable DNA product. The use of a thermolabile UDG is important, as complete inactivation of the UDG is required to prevent destruction of newly synthesized qPCR products.

     To enable carryover prevention, 0.025 units/μl Antarctic Thermolabile UDG (NEB #M0372) should be added to the reaction mix. To maximize elimination of contaminating products, set up the qPCR experiments at room temperature or include a 10 minute incubation step at 25°C before the initial denaturation step.

  8. Reaction Setup and Cycling Conditions
     Due to dual hot-start feature of Luna One-Step Kits, it is not necessary to set up reactions on ice or preheat the thermocycler prior to use.
     For 96-well plates, a final reaction volume of 20 μl is recommended.
     For 384-well plates, a final reaction volume of 10 μl is recommended.
     When programming instrument cycling conditions, ensure a plate read is included at the end of the extension step, and a denaturation (melt) curve after cycling is complete to analyze product specificity.
     Amplification for 40 cycles is sufficient for most applications, but for very low input samples 45 cycles may be used.

操作说明、说明书 & 用法

• 操作说明

  1. Luna^® Probe One-Step RT-qPCR Kit (No ROX) Protocol (NEB# E3007)

• 说明书

  产品说明书包含产品使用的详细信息、产品配方和质控分析。

  • manualE3007

• 应用实例

  • High-throughput qPCR and RT-qPCR Workflows Enabled by Beckman Coulter Echo Acoustic Liquid Handling and NEB Luna Reagents
  • Facilitating Detection of SARS-CoV-2 Directly from Patient Samples: Precursor Studies with RT-qPCR and Colorimetric RT-LAMP 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. What is the difference between probe- and dye-based versions of the Luna^® qPCR Mixes?
  4. Should I use probe- or dye-based detection for my qPCR assays?
  5. How should I design primers for Luna^® qPCR?
  6. How long should my amplicon be for qPCR?
  7. Why is the Luna^® qPCR Mix blue? Will this dye interfere with detection?
  8. Can I use fast instrument settings with the Luna^® qPCR Mix?
  9. Do I need to add ROX?
  10. How many dilutions should I use to make a standard curve?
  11. Why does NEB recommend 40-45 cycles?
  12. Does the Luna^® qPCR Mix contain dUTP? Can I use carryover contamination prevention methods?
  13. Can I run multiplex reactions with the Luna^® Probe One-Step RT-qPCR Kits? Do I need to change my reaction conditions?
  14. Can I use a ROX-labeled probe with the Luna^® Probe Mixes that contain a universal ROX reference dye?
  15. Can alternative probe based detection strategies be used with the Luna^® Probe Mix?
  16. How much primer and probe should I use with the Luna^® Universal Probe RT-qPCR Kit?
  17. How do I choose between one-step RT-qPCR and two-step RT-qPCR?
  18. What RNA samples can be used in RT-qPCR with the Luna^® Mix?
  19. How much RNA template should I use in my RT-qPCR reaction?
  20. Can I use longer targets in one-step RT-qPCR?
  21. What temperature should I use for cDNA synthesis with Luna^® RT-qPCR kits?
  22. Should I include a no Luna^® WarmStart^® Enyzme Mix control (-RT Control)?
  23. Is the Monarch Total RNA Miniprep Kit compatible with Luna RT-qPCR Reagents?
  24. Are the Monarch RNA Cleanup Kits (NEB # T2030, #T2040, #T2050) compatible with Luna RT-qPCR reagents?
  25. Can I use shorter cycling times?
  26. What are the differences between the Luna One-Step RT-qPCR products (NEB #E3006, #E3007, #M3019, #M3029 and #L4001)?
  27. Can I run the Luna^® qPCR Mix on my qPCR instrument?
  28. Can I use the Luna Probe RT-qPCR mixes for viral detection?