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 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. 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.
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.
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Nucleic acid-based aptamers for use with thermophilic DNA polymerases and reverse transcriptases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the Luna® Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) product for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact firstname.lastname@example.org.
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