Intended for RT-PCR, the LunaScript Multiplex One-Step RT-PCR Kit offers a streamlined protocol for cDNA synthesis and PCR amplification in a single reaction. The 5X reaction mix contains dNTPs and is optimized for multiple target detection in a simple workflow. The 25X enzyme mix features Luna WarmStart Reverse Transcriptase and Q5 Hot Start High-Fidelity DNA Polymerase. The dual-temperature control of enzyme activities by aptamer-based inhibition enables room temperature reaction setup, with preassembled reactions stable at room temperature for up to 24 hours. The kit has robust multiplex target amplification capacity and enables various applications such as diagnostics, pathogen detection, and viral genome sequencing (including the ~50 amplicons per reaction used in ARTIC SARS-CoV-2 sequencing protocols).Multiplex RNA target detection and identification from a single RT-PCR reactionThe LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555) requires only RNA template and gene-specific primers to enable multiplex cDNA target synthesis and amplification in a single reaction. Amplified cDNA products can be detected or identified by downstream applications including next-generation sequencing, DNA arrays, fragment analysis, electrophoresis, and traditional cloning/sequencing workflows.Superior multiplexing and sensitivity with the LunaScript Multiplex One-Step RT-PCR KitHuman spleen total RNA was used to amplify 7 targets ranging from 185–776 bp using the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555) or the SuperScript IV One-Step RT-PCR System (Thermo Fisher Scientific). 40% of each RT PCR experiment was loaded onto a 2% agarose gel. For LunaScript reactions, the standard protocol of 1 μM final primer concentration and a 1 minute annealing step was used. For the SSIV reactions, the recommended conditions failed to amplify all bands. Additional conditions were tested, varying key parameters of primer concentration and annealing time. The LunaScript Multiplex One-Step RT-PCR Kit successfully amplified all 7 targets from a variety of RNA inputs, and no conditions were identified that enabled the SuperScript IV One-Step RT-PCR System to amplify the full spectrum of targets, even at high input.The LunaScript Multiplex One-Step RT-PCR Kit enables simple ARTIC SARS-CoV-2 sequencing workflowsAmplicons were generated from 1000 copies of SARS-CoV-2 viral gRNA (ATCC VR-1986) in 100 ng of Universal Human Reference RNA (Thermo Fisher Scientific®, QS0639) using LunaScript Multiplex One-Step RT-PCR Kit with ARTIC V3 primers from either NEB (A) or IDT (B). cDNA synthesis was conducted in duplicate 12.5 μl reactions for each primer pool (1 and 2) using the following cycling conditions: 20 min at 55°C (reverse transcription), 1 min at 98°C (RT inactivation/initial denaturation), followed by 35 cycles of 15 sec at 95°C and 5 min at 63°C (annealing and extension).Libraries were constructed from 1.3 μl of each sample of pooled amplicons using the NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) and sequenced on a MiSeq® instrument (2X75 bp). Sequencing reads per amplicon were plotted for each library. Reads were downsampled with seqtk (250,000 read pairs) and aligned to the SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie 2. Reference line indicates typical quality threshold of 100 reads.Sensitive and robust detection of RNA with up to 24 hours of room temperature stabilityA 570 bp GAPDH target was amplified from 10 ng down to 0.01 pg of Jurkat total RNA using the LunaScript Multiplex One-Step RT-PCR Kit (NEB #E1555). Reactions were set up on ice and run immediately (Control) or were set up and left at room temperature for 1, 4 or 24 hours before thermocycling. No template control (NTC) reactions were set up for each incubation condition. Highly sensitive and specific target amplification was observed at all input amounts, even after the preassembled reactions were left at room temperature for up to 24 hours.Comparing the LunaScript and OneTaq one-step RT-PCR products
This product is related to the following categories:
RT-PCR Products,
Reverse Transcriptases & RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products
This product can be used in the following applications:
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