LyoPrime Luna® Probe One-Step RT-qPCR Mix with UDG
Supplied as a lyophilized cake, the LyoPrime Luna® Probe One-Step RT-qPCR Mix with UDG enables sensitive detection of target RNA sequences in a room temperature-stable format. This product contains the same versatile features and strong performance as the liquid version: Luna® Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019).
The LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG is offered in a lyophilized format, allowing for ambient/room temperature shipping and storage prior to use. It is designed for real-time detection of target RNA sequences using hydrolysis probes. Performance in multiplexing applications has been optimized, with sensitive, linear detection of up to 5 targets across a range of inputs. The stable cake can be resuspended to different concentrations to accommodate a variety of sample input volumes. It is available in 2 x 60 reaction glass vials or a 96-well plate pack size (1 reaction per well). These products can be customized (e.g. with the addition of primers and probes) – please contact us for more information.
Figure 1: LyoPrime Luna offers a robust, versatile RT-qPCR option in a shelf-stable lyophilized format
The LyoPrime Luna® RT-qPCR Mix (NEB #L4001) offers the same versatile features as the Luna RT-qPCR 4X Mix (NEB #M3019) in a lyophilized format that can be shipped and stored at room temperature. It is available in glass vials or a 96-well plate.
The mix contains the necessary components for one-step RT-qPCR, 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 WarmStart-activated reverse transcriptase allows for dual control of enzyme activity via reversible, aptamer-based inhibition. This temperature-dependent activation helps prevent undesirable non-specific priming and extension prior to thermocycling, enabling room temperature reaction set-up after rehydration. 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.
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 a product of a previous amplification can serve as the substrate of a subsequent reaction. Unlike E.coli UDG, 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 avoid pipetting errors. The tracking dye does not overlap spectrally with fluorophores commonly used in qPCR and does not interfere with real-time detection.
To learn about the lyophilization process, or to inquire about customizing this product to meet your assay needs, please visit www.neb.com/lyoprime.
Figure 2: Lyophilized and liquid Luna RT-qPCR mixes demonstrate equivalent strong performance
A. RT-qPCR targeting human β-actin was performed using either the LyoPrime Luna® Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) or Luna® Probe One-Step 4X Mix with UDG (NEB #M3019) over an 8-log range of input template concentrations (1 μg – 0.1 pg Jurkat total RNA) with 4 replicates at each concentration, run on an ABI® QuantStudio™ 6 Flex real-time instrument. Reactions (20 μl) included primers at 400 nM each and and probe at 200 nM, and followed the product recommended cycling conditions. B. Standard Curve results were substantially equivalent for the lyophilized (gold) and liquid-format (blue) mixes, with strong linearity and reproducibility observed, even at the lowest concentrations tested.
Figure 3: LyoPrime Luna enables robust multiplexing
Multiplex RT-qPCR was performed using the LyoPrime Luna® Probe One-Step RT-qPCR Mix with UDG over a 6-log range of Jurkat total RNA (1 μg 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: LyoPrime Luna matches the performance of the liquid Luna reagent and outperforms other commercial lyophilized mixes
Commercially-available lyophilized RT-qPCR reagents were tested on 8 human RNA targets varying in abundance, length, and %GC. Data was collected by 2 users and according to manufacturers’ 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). The LyoPrime Luna Probe RT-qPCR Mix with UDG (NEB #L4001) outperformed other lyophilized products, with strong results comparable to those of the liquid format Luna Probe RT-qPCR Mix with UDG (NEB #M3019).
Learn more about our comprehensive qPCR/RT-qPCR testing and “dots in boxes” data visualization here.
Figure 5: Lyophilized and liquid Luna reagents demonstrate sensitive detection of SARS-CoV-2 viral RNA
Multiplex RT-qPCR was performed using primers and probes from the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit (NEB #E3019) targeting 2019-nCoV_N1 (N1, HEX™), 2019-nCoV_N2 (N2, FAM™) and human RNase P (RP, Cy5), run on a Bio-Rad® CFX384 real-time instrument. Reactions (5 μl) included primers at 400 nM each and and probes at 200 nM each, and followed the product recommended cycling conditions. Performance was evaluated using Synthetic Twist SARS-CoV-2 RNA Control 2 diluted in 5 ng/μl human Jurkat total RNA. The Luna and LyoPrime Luna RT-qPCR Mixes both exhibited linear quantitation (standard curves of 100,000 to 10 copies SARS-CoV-2 RNA, left) and sensitive detection (100% detection of N1 and N2 with 40 replicates at 5 and 10 copies SARS-CoV-2 RNA; no false positive detection with 28 negative control replicates). Similar results were obtained for 20 μl reactions in 96-well format.
Figure 6: LyoPrime Luna reagents enable strong detection and quantitation of various infectious disease targets
Duplex RT-qPCR was performed using primers and probes targeting the indicated viral RNA gene (FAM) and human RNase P (Cy5) on a Bio-Rad CFX96 instrument. Performance was evaluated for triplicate standard curves of 100,000 to 10 copies ATCC® synthetic viral RNA diluted in 5 ng/μl human (Jurkat) total RNA (Chikungunya Virus: VR-3246SD; Dengue-2 Virus: VR-3229SD; Zika Virus: VR-3252SD). For each target the corresponding ATCC reference primer and probe sequences were used. Reactions (20 μl) included primers at 400 nM each and probes at 200 nM and followed the product recommended cycling conditions. The LyoPrime Luna RT-qPCR Mix exhibited linear quantitation and sensitive detection for each target.
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Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Licenses
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 LyoPrime Luna™ Probe One-Step RT-qPCR Mix with UDG 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 busdev@neb.com.
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