Rapid, sensitive and precise probe-based qPCR detection and quantitation of target DNA and cDNA sequences.
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 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 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: NEB’s 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 NEB Protoscript® II First Strand cDNA Synthesis Kit ( NEB #E6560).
Figure 2: NEB’s 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 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: 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. NEB’s 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