The Luna Probe One-Step RT-qPCR 4X Mix with UDG 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.
Due to the one-step RT-qPCR workflow and probe-based detection chemistry, a comparison can be made to the Luna Probe One-Step RT-qPCR Kit (NEB #E3006).
Figure 1: A new product choice for one-step RT-qPCR assays
The Luna Probe One-Step RT-qPCR Mix with UDG 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 Taq DNA 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. Additionally, the mix is 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.
Figure 2: Robust amplification and detection of different viral RNA with Luna Probe One-Step RT-qPCR 4X Mix with UDG
RT-qPCR targeting SARS-CoV-2 (N1 target) and H. influenza H1N1 (HA target) was performed using the Luna Probe One-Step RT-qPCR Mix with UDG. Performance in 20 μl reactions was evaluated in two real-time instruments over a 5-log range of (A) 10–100,000 copies Synthetic SARS-CoV-2 RNA Control 2 (Twist Biosciences, SKU: 102024) diluted in 10 ng of Jurkat total RNA (BioChain, #R1255815-50) and (B) 12.4–124,000 copies Influenza A (H1N1) RNA (ATCC®VR-95DQ™) diluted in 10 ng Jurkat total RNA. Sensitive, linear performance can be observed in the amplification of both viral targets.
Figure 3: Multiplex detection (5 targets) with the Luna Probe One-Step RT-qPCR 4X Mix with UDG
Multiplex RT-qPCR was performed using the Luna Probe One-Step RT-qPCR 4X Mix with UDG 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 and efficiencies for each of the 5 human targets are shown. 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 strong efficiency and R2 values.
Figure 4: The Luna Probe One-Step RT-qPCR 4X Mix with UDG outperforms comparable products
One-step RT-qPCR was tested on 8 RT-qPCR targets (indicated by color) varying in abundance, length, and %GC. Data was collected on multiple days by two users according to manufacturer’s recommendations using the Applied Biosystems™ QuantStudio™ 6 real-time PCR system. 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). Although both products performed reasonably well, NEB’s Luna Probe RT-qPCR 4X Mix with UDG outperformed the TaqPath 1-Step RT-qPCR Master Mix, CG, as evidenced by the higher number of experiments whose results fell in the green box.
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 5: Evaluation of RT-qPCR carryover prevention
To evaluate the capacity of carryover product digestion, a uracil-containing RT-qPCR product was generated using the Luna One-Step Probe RT-qPCR kit. Approximately 105 copies of the uracil-containing product were used as template for subsequent qPCR reactions using three different RT-qPCR master mixes (all containing UDG). Following manufacturer’s recommended protocols, incubation at 25˚C at the start of the 2nd reaction enables UDG to degrade the dU-containing input, reducing its ability to contribute to a (false) positive signal. The ΔCq value is the cycle difference between carryover treatment and no carryover treatment of the same input. Larger ΔCq values indicate more efficient carryover product digestion. After decontamination using the Luna mix, full product digestion was observed in one sample and a large ΔCq was observed in the second. Note that the amount of DNA present in true contamination events is difficult to assess/predict.
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.
The Luna Probe One-Step RT-qPCR Master Mix with UDG contains an inert blue tracking dye to eliminate pipetting errors.
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