NEB Luna Universal One-Step RT-qPCR Kit (Cat No. E3005)

Rapid, sensitive and precise dye-based qPCR detection and quantitation of target RNA targets

Make a simpler choice

• One product per application simplifies selection
• Convenient master mix formats and user-friendly protocols simplify reaction setup
• Non-interfering, visible tracking dye helps to eliminate pipetting errors

Experience best-in-class performance

• All Luna^® products have undergone rigorous testing to optimize specificity, sensitivity, accuracy and reproducibility
• Products perform consistently across a wide variety of sample sources
• A comprehensive evaluation of commercially-available qPCR and RT-qPCR reagents demonstrates superior performance of Luna products

Optimize your RT-qPCR with Luna WarmStart^® Reverse Transcriptase

• Novel, thermostable reverse transcriptase (RT) improves performance
• WarmStart RT paired with Hot Start Taq increases reaction specificity and robustness

Product Information

Rapid, sensitive and precise dye-based qPCR detection and quantitation of RNA targets.

The Luna Universal One-Step RT-qPCR Kit is optimized for dye-based real-time quantitation of target RNA sequences via the SYBR^®/FAM fluorescence channel of most real-time instruments. Dye-based qPCR/RT-qPCR uses real-time fluorescence of a double-stranded DNA (dsDNA) binding dye, most commonly SYBR Green I, to measure DNA amplification after each PCR cycle. At a point where the fluorescence signal is confidently detected over the background fluorescence, a quantification cycle or C_q value can be determined. C_q values can be used to evaluate relative target abundance between two or more samples, or to calculate absolute target quantities in reference to an appropriate standard curve derived from a series of known dilutions.

One-Step RT-qPCR provides a convenient and powerful method for RNA detection and quantitation. In a single tube, RNA is first converted to cDNA by a reverse transcriptase, and then a DNA-dependent DNA polymerase amplifies the cDNA, enabling quantitation via qPCR.

In the Luna Universal One-Step RT-qPCR Kit, Hot Start Taq DNA Polymerase is combined with a novel WarmStart-activated reverse transcriptase, allowing 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 Reverse Transcriptase also possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C. For difficult targets/templates, higher RT step temperatures of up to 60°C can be used without compromising Luna performance.

Note that to ensure full activation of the Luna WarmStart RT, incubation at temperatures lower than 50°C is not recommended.
 
The Luna Universal One-Step Reaction Mix is supplied at 2X concentration and contains Hot-Start Taq DNA Polymerase, dNTPs, a fluorescent dsDNA-binding dye, and all required buffer components. It 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. The Reaction Mix also features dUTP for carryover prevention and a non-fluorescent visible dye for monitoring reaction setup. This visible dye does not overlap spectrally with the included dsDNA-binding dye and does not interfere with real-time detection.

The Luna WarmStart RT Enzyme Mix is supplied at 20X concentration and contains Luna WarmStart Reverse Transcriptase as well as Murine RNase Inhibitor to aid in preventing RNA degradation (see also template preparation in product manual ). It is compatible with various RNA sample types (total RNA, poly(A)-RNA, etc.) and sources.

Figure 1: The Luna Universal One-Step RT-qPCR Kit offers exceptional sensitivity, reproducibility and RT-qPCR performance

Figure 2: The Luna Universal One-Step RT-qPCR Kit provides sensitive and accurate detection and quantitation across a wide variety of RNA sources

Figure 3: Extensive performance evaluation of commercially available dye-based RT-qPCR reagents demonstrates the robustness and specificity of Luna

• • Product Notes

    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.

    Primer Concentration
    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.

    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.

    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.

    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.

    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.

    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.

• Protocols, Manuals & Usage

  Protocols

  1. Luna^® Universal One-Step RT-qPCR Kit Protocol (E3005)
     
     
     

  Manuals

  The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

  • manualE3005
    
    
    

  Usage Guidelines

  • Optimization Tips for Luna® One-Step RT-qPCR

• • 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 run the Luna^® qPCR Mix on my qPCR instrument?
    9. Can I use fast instrument settings with the Luna^® qPCR Mix?
    10. Do I need to add ROX?
    11. How many dilutions should I use to make a standard curve?
    12. Why does NEB recommend 40-45 cycles?
    13. Does the Luna^® qPCR Mix contain dUTP? Can I use carryover contamination prevention methods?
    14. Why do I have multiple peaks in my melt curve?
    15. How can I distinguish non-template amplification (NTC) from real products?
    16. Why do I see amplification curves in my NTC samples?
    17. How much primer should I use with the Luna^® Universal RT-qPCR Kit?
    18. How do I choose between one-step RT-qPCR and two-step RT-qPCR?
    19. What RNA samples can be used in RT-qPCR with the Luna^® Mix?
    20. How much RNA template should I use in my RT-qPCR reaction?
    21. Can I use longer targets in one-step RT-qPCR?
    22. What temperature should I use for cDNA synthesis with Luna^® RT-qPCR kits?
    23. Should I include a no Luna^® WarmStart^® Enyzme Mix control (-RT Control)?
    24. What is the fluorescent, double-stranded DNA binding dye in the Luna^® qPCR master mix?
    25. Is the Monarch Total RNA Miniprep Kit compatible with Luna RT-qPCR Reagents?
    26. Are the Monarch RNA Cleanup Kits (NEB # T2030, #T2040, #T2050) compatible with Luna RT-qPCR reagents?
    27. Can I use shorter cycling times?

  • Troubleshooting

    • Luna® One-Step RT-qPCR Troubleshooting Guide