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 performanceqPCR 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 applicationsSingleplex (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 LunaqPCR 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 visualizationFigure 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:
Luna? qPCR & RT-qPCR Products,
PCR, qPCR & Amplification Technologies Products
This product can be used in the following applications:
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