Description:

 

Rapid, sensitive and precise dye-based qPCR detection and quantitation of target DNA and cDNA sequences.

Dye-based quantitative PCR (qPCR) uses real-time fluorescence of a double-stranded DNA (dsDNA) binding dye, most commonly SYBR^® Green I, to measure DNA amplification during each cycle of a PCR. 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.The NEB Luna Universal qPCR Master Mix is an optimized 2X reaction mix for real-time qPCR detection and quantitation of target DNA sequences using the SYBR^®/FAM channel of most real-time qPCR instruments. 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 with fluorescent dyes 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 and DNA template. Genomic DNA or cDNA of interest can be quantitated with Luna qPCR, and existing as well as commercial qPCR assay primer sequences can be used.Figure 1: NEB’s Luna Universal qPCR Master Mix offers exceptionalsensitivity, reproducibility and qPCR performanceqPCR targeting human GAPDH was performed using the Luna Universal 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 NEB Protoscript® II First Strand cDNA Synthesis Kit (NEB #E6560).Figure 2: NEB’s Luna Universal qPCR Master Mix provides sensitive and accuratedetection and quantitation across a wide variety of DNA sourcesThe Luna Universal qPCR Master Mix is compatible with a broad range of genomic DNA sources. qPCR targets were quantitated with 50 ng – 0.5 pg genomic DNA as input using an ABI 7500 Fast real-time instrument. Genomic DNA was purified by typical column-based methods. In these examples, strong performance can be observed in the amplification of ACTB (encoding β-actin) from Mouse kidney genomic DNA, psbB (Photosystem II CP47 reaction center protein PsbB) from Tobacco, and RDN18 (18S ribosomal RNA) from Yeast.Figure 3: Extensive performance evaluation of commercially available dye-based qPCR reagents demonstrates the robustness and specificity of LunaqPCR reagents from NEB and other manufacturers were tested across 16–18 qPCR targets varyingin abundance, length and %GC, using either Jurkat genomic DNA or Jurkat-derived cDNA as input (10 genomic DNA targets and 8 cDNA targets on Bio-Rad real-time instrument, 9 genomic and 7 cDNA targets on ABI instrument). For each testing condition, data was collected by 2 users and according to manufacturer’s specifications. Results were evaluated for efficiency, low input detection and lack of non-template amplification (where ΔC_q = average C_q of non-template control – average C_q 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: Bio-Rad, SsoAdvanced™ Universal SYBR® Green Supermix; Roche, FastStart™ SYBR Green Master; QIAGEN, QuantiTect^® SYBR Green PCR Kit; ABI, PowerUP™ SYBR Green Master Mix; Promega^®, GoTaq^® qPCR Master Mix. NEB’s Luna Universal qPCR Master Mix outperformed all other reagents tested.Learn more about our comprehensive qPCR/RT-qPCR testing and \"dots in boxes” data visualization

Notes:

Primer DesignThe 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 ConcentrationFor most targets, a final concentration of 250 nM (each primer) will provide optimum performance. If needed, primer concentrations can be optimized between 100–500 nM.Amplicon LengthTo 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 ConcentrationLuna 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 before addition to qPCR.ROX Reference DyeSome real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. The Luna Universal qPCR Master Mix is formulated with a universal reference dye that is compatible with a variety of qPCR instrument types, including those that use no passive reference normalization and those that use a low or high concentration of passive reference dye (ROX). Therefore, no additional components are required to ensure compatibility with these instruments.Carryover Contamination PreventionqPCR 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 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 ConditionsDue 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.