The Luna Probe One-Step RT-qPCR Kit (No ROX) is optimized for real-time quantitation of target RNA sequences using hydrolysis probes. 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. Probe-based qPCR/RT-qPCR monitors an increase in fluorescence 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 confidently detected 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.
In the Luna Probe One-Step RT-qPCR Kit (No ROX), 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 WarmStart Luna 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 WarmStart Luna RT, incubation at temperatures lower than 50°C is not recommended.
The Luna Probe One-Step RT-qPCR Kit (No ROX) is supplied at 2X concentration and contains Hot-Start Taq DNA Polymerase, dNTPs, and all required buffer components. This formulation contains no reference dye and is compatible with any instrument that does not require ROX (if ROX normalization is needed, ROX can be added.) The Reaction Mix features dUTP for carryover prevention and a non-fluorescent visible dye for monitoring reaction setup. This visible dye does not overlap spectrally with fluorophores commonly used in qPCR 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.
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Figure 1: NEB’s Luna Universal Probe One-Step RT-qPCR Kit* offers exceptional sensitivity, reproducibility and RT-qPCR performance RT-qPCR targeting human GAPDH was performed using the Luna Universal Probe One-Step RT-qPCR Kit over an 8-log range of input template concentrations (1 μg – 0.1 pg Jurkat total RNA) with 8 replicates at each concentration. Reaction setup and cycling conditions followed recommended protocols, including a 10-minute RT step at 55°C for the thermostable Luna WarmStart Reverse Transcriptase.
Figure 2: NEB’s Luna Universal Probe One-Step RT-qPCR Kit* offers robust performance in multiplex applications Multiplex RT-qPCR targeting human GAPDH, ribosomal protein L32g and PI3-Kinase-Related Kinase SMG1 was performed using the Luna Universal Probe One-Step RT-qPCR Kit* over a 7-log range of input template concentrations (1 μg – 1 pg Jurkat total RNA) with 4 replicates at each concentration. Amplification plots are shown both overlayed (left) and for each multiplex target (right). To account for copy number differences, 0.4 µM primer was used for lower-copy target (SMG1) and 0.2 µM primer for higher-copy targets (L32g and GAPDH). Luna maintains superior efficiency, reproducibility, sensitivity and performance in multiplex RT-qPCR.
Figure 3: Extensive performance evaluation of commercially available probe-based RT-qPCR reagents demonstrates the robustness and specificity of Luna Commercially-available RT-qPCR reagents were tested on 7 RT-qPCR targets varying in abundance, length, and %GC. Data was collected by 2 users and according to manufacturer’s recommendations. 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 suppliers are shown: Quanta, qScript™ XLT 1-Step RT-qPCR ToughMix®; ABI, TaqMan® RNA-to-Ct 1-Step Kit; QIAGEN, QuantiFast® Probe RT-PCR Kit; Bio-Rad, iTaq™ Universal Probes One-Step Kit; Promega®, GoTaq® Probe 1-Step RT-qPCR System. NEB’s Luna Universal Probe One-Step RT-qPCR Kit* outperformed all other reagents tested.
Learn more about our comprehensive qPCR/RT-qPCR testing and “dots in boxes” data visualization.
* Data was collected using the Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006). Luna Probe One-Step RT-qPCR Kit (NO ROX) (NEB #E3007) offers similar performance.
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