Probe-based quantitative PCR (qPCR) uses real-time fluorescence released upon 5´→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 significantly detectable 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 or to calculate absolute target quantities in reference to an appropriate standard curve, derived from a series of known dilutions.
The Luna Universal Probe qPCR Master Mix is a 2X reaction mix optimized for real-time qPCR detection and quantitation of target DNA sequences using hydrolysis probes. It contains Hot Start Taq DNA Polymerase and has been 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). It also features dUTP for carryover prevention and a non-fluorescent, visible dye to monitor reaction setup. This dye does not spectrally overlap fluorophores commonly used for qPCR and will not interfere with real-time detection.
The master mix formulation is supplied at 2X concentration and contains all PCR components required for amplification and quantitation of DNA except primers/probes and DNA template. Genomic DNA or cDNA of interest can be quantitated with Luna qPCR and existing as well as commercial qPCR assay primer/probe sequences can be used.
Figure 1: The Luna Universal Probe qPCR Master Mix offers exceptional sensitivity, reproducibility and qPCR performance qPCR targeting human GAPDH was performed using the Luna Universal Probe qPCR Master Mix over a 6-log range of input template concentrations (20 ng – 0.2 pg Jurkat-derived cDNA) with 8 replicates at each concentration. cDNA was generated from Jurkat total RNA using the Protoscript® II First Strand cDNA Synthesis Kit (NEB #E6560).
Figure 2: The Luna Universal Probe qPCR Master Mix offers robust performance in multiplex applications Singleplex (left) and multiplex (right) qPCRs targeting human GAPDH, ribosomal protein L32g and PI-3-Kinase-Related Kinase SMG1 were performed using the Luna Universal Probe qPCR Master Mix over a 5-log range of input template concentrations (20 ng – 2 pg Jurkat-derived cDNA) with 4 replicates at each concentration. 0.4 µM primer was used for the lower-copy target (SMG1) and 0.2 µM primer for each higher-copy target (L32g and GAPDH), in both multiplex qPCR (to account for copy number differences) and singleplex qPCR (to allow direct comparison).
Figure 3: Extensive performance evaluation of commercially available probe-based qPCR reagents demonstrates the robustness and specificity of Luna qPCR reagents from NEB and other manufacturers were tested on 10 qPCR targets, varying in abundance, length and % GC, using either Jurkat genomic DNA or Jurkat-derived cDNA as input (5 targets each). For each testing condition, data was collected by 2 users and according to manufacturer specifications. 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 (Quality Score). Bar graph indicates % of targets that met acceptable performance criteria (indicated by green box on dot plot and Quality Score > 3). Results for NEB and other major manufacturers are shown: QIAGEN, QuantiTect® Probe PCR Kit; Bio-Rad, SsoAdvanced™ Universal Probes Supermix; Roche, FastStart® TaqMan® Probe Master; ABI, TaqMan Fast Advanced Master Mix; Promega®, GoTaq® Probe qPCR Master Mix. The Luna Universal Probe qPCR Master Mix outperformed all other reagents tested.
Learn more about our comprehensive qPCR/RT-qPCR testing and “dots in boxes” data visualization
Figure 4: The Luna Universal Probe qPCR Master Mix contains an inert blue tracking dye to eliminate pipetting errors.
This product is related to the following categories:
Assay 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 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). For cDNA targets, it is advisable to design primers across known splicing sites in order to prevent amplification from genomic DNA. Conversely, primers designed to target intronic regions can ensure amplification exclusively from genomic DNA.
Primer and Probe Concentration For most targets, a final concentration of 400 nM for each primer will provide optimum performance. If needed, primer concentrations can be optimized between 200–900 nM. Probe should be included at 200 nM for best results. Probe concentration can be optimized in the range of 100–500 nM if optimization of performance or target fluorescence level is desired.
Multiplexing To detect or quantitate multiple targets in the same Luna reaction, select different fluorophores corresponding to separate detection channels of the real-time instrument. Include 400 nM of forward and reverse primer and 200 nM probe for each target to be detected in the reaction, and adjust concentrations if necessary based on performance (primer 200–900 nM, probe 100–500 nM). When loading 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. For ROX-dependent instruments, avoid ROX-labeled probes. The functionality of the primer and probe sets should be tested individually before attempting a multiplex reaction. When determining which fluorophores to include in a multiplex reaction, be sure to choose compatible reporter dyes and quenchers that have well separated fluorescence spectra or exhibit minimal overlap.
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 (including the use of longer extension times), for targets that exceed that range.
Template Preparation and Concentration Luna qPCR is compatible with DNA samples prepared through typical nucleic acid purification methods. Prepared DNA 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 by dilution into either TE or water. Generally, a useful concentration of standard and unknown material will be in the range of 106 copies to 1 copy. For gDNA samples from large genomes (e.g., human, mouse) a range of 50 ng–1 pg of gDNA is typical. For small genomes, adjust as necessary using 106 –1 copy input as an approximate range. Note that for single copy dilutions, some samples will contain multiple copies and some will have none, as defined by the Poisson distribution. For cDNA, use the product of a reaction containing 1 μg–0.1 pg starting RNA. cDNA does not need to be purified before addition to the Luna reaction but should be diluted at least 1:10 into the qPCR.
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 qPCR is an extremely sensitive method, and contamination in new 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, the Luna Universal Probe qPCR Master Mix contains a mixture of dUTP/dTTP that results in the incorporation of dU into the DNA product during amplification. Pretreatment of 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 the hot start nature of the polymerase, it is not necessary to preheat the thermocycler prior to use or set up reactions on ice. For 96-well plates, we recommend a final reaction volume of 20 μl. 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.
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 & Change Notifications
Effective May 2, 2022, a secondary bottle supplier has been qualified. The alternative 30 mL bottle has a different cap color and may be used for relevant pack sizes with no change in product performance or stability.
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]
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]
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 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 Universal Probe qPCR Master Mix 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.
Trademarks
BIO-RAD® is a registered trademark of Bio-Rad Laboratories. FASTSTART™ is a trademark of Roche. GOTAQ® and PROMEGA® are registered trademarks of Promega Corporation. QIAGEN® and QUANTITECT® are registered trademarks of Qiagen, Inc. QUANTSTUDIO® is a registered trademark of Life Technologies Corporation. SOMAMER® is a registered trademark of Somalogic, Inc. SSOADVANCED™ is a trademark of Bio-Rad Laboratories. STEPONE® is a registered trademark of Applied Biosystems, LLC. SYBR® is a registered trademark of Molecular Probes, Inc. TAQMAN® is a registered trademark of Thermo Fisher Scientific.
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