The Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) is designed for real-time detection of target RNA sequences using hydrolysis probes. In a single tube, RNA is first converted to cDNA by a reverse transcriptase, then a DNA-dependent DNA polymerase amplifies the cDNA, enabling quantitation via real time or quantitative PCR (qPCR). Probe-based qPCR/RT-qPCR monitors an increase in fluorescence upon 5´ to 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.The Luna Probe One-Step RT-qPCR Mix with UDG (No ROX) is supplied at a 4X concentration and enables higher amounts of sample input, which is relevant for applications where RNA present in low abundance is of interest, such as pathogen detection. Performance in multiplexing applications has been optimized, with sensitive, linear detection achieved for up to 5 targets across a range of inputs. The mix consolidates the necessary components for one-step RT-qPCR into a single tube, including Luna WarmStart RT, Hot Start Taq DNA Polymerase, dNTPs, and Murine RNase Inhibitor in an optimized buffer. Combining Hot Start TaqDNA Polymerase with a novel, WarmStart-activated reverse transcriptase allows for 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 Luna WarmStart RT also possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C. 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 sensitivity of RT-qPCR makes it important to minimize DNA contamination wherever possible. The inclusion of dUTP and thermolabile UDG prevents carryover contamination, where unintended product of a previous amplification can serve as the substrate of a subsequent reaction. The thermolabile UDG is completely inactivated at temperatures above 50°C, thereby having no effect on qPCR performance.
Figure 1: Detection of RNA viruses using the Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX)RT-qPCR targeting SARS-CoV-2 (N1 target), Zika (POLY target), Dengue (E target), Chikungunya (E2 glycoprotein target) was performed using the Luna Probe One-Step RT-qPCR Mix with UDG (No ROX). Performance in 20 μl reactions was evaluated on a Bio-Rad® CFX96 real-time instrument over a 5-log range of template (100,000–10 copies/reaction). Templates including Synthetic SARS-CoV-2 RNA Control 2 (Twist Biosciences, SKU: 102024), Synthetic Zika virus (ZIKV) RNA (ATCC® VR-3252SD™), Synthetic RNA from Dengue virus type 2 (ATCC VR-3229SD™), Synthetic RNA from Chikungunya virus (ATCC VR-3246SD™), were all individually diluted in 10 ng of Jurkat total RNA (BioChain, #R1255815-50). Sensitive, linear performance can be observed in the amplification of all viral targets.Figure 2: Consistent multiplex amplification using the Luna Probe One-Step RT-qPCR 4X Mix with UDG, with and without ROX universal reference dyeMultiplex RT-qPCR was performed using the Luna Probe One-Step RT-qPCR 4X Mix with UDG with and without ROX universal reference dye (NEB #M3019 and NEB #M3029) over a 5-log range of Jurkat total RNA (100 ng to 10 pg) on a Bio-Rad CFX96 real-time instrument. Amplification standard curves of 5 human targets are shown for both Luna products. An overlay of standard curves for the ACTIN target (which includes a Texas Red labeled probe) comparing the Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) and the Luna Probe One-Step RT-qPCR 4X Mix with UDG demonstrates robust performance without interference from the ROX universal reference dye. Reactions (20 μl) included primers and probes at 200 nM each and followed the product recommended cycling conditions. All five targets were detected linearly in the multiplex reactions with robust efficiency and R2 values as shown in C.Figure 3:High quality results between operators using the Luna Probe One-Step RT-qPCR 4X Mix with UDG, with and without ROX universal reference dyeOne-step RT-qPCR was tested on 8 RT-qPCR targets (indicated by color) varying in abundance, length, and %GC. Data was collected by two users according to manufacturer’s recommendations using the Bio-Rad CFX96 real-time instrument. 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 based on metrics described previously (Quality Score). The Luna Probe RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029) shows comparable performance to the Luna Probe RT-qPCR 4X Mix with UDG (NEB #M3019) as evidenced by the same number of experimental results that fell in the green box.A non-fluorescent visible blue tracking dye is included to assist in pipetting into clear vessels. The tracking dye does not overlap spectrally with fluorophores commonly used in qPCR and does not interfere with real-time detection.Figure 4:The Luna Probe One-Step RT-qPCR Master Mix with UDG (No ROX) 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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