Product Class: Kit

LunaScript® Multiplex One-Step RT-PCR Kit


Catalog #E1555

Product Introduction

 

Product Information

Description

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 reaction



The 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 Kit



Human 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 workflows



Amplicons 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 stability



A 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,
cDNA Synthesis & Reverse Transcriptases Products,
This product can be used in the following applications:
RT-PCR,
cDNA Synthesis,
RT-PCR & cDNA Synthesis

Properties & Usage

Features

  • Streamlined one-step RT-PCR protocol with Luna WarmStart Reverse Transcriptase and Q5 Hot Start High-Fidelity DNA Polymerase
  • High sensitivity: detect as low as 0.01 pg of human total RNA
  • Superior multiplexing capacity
  • Up to 24 hours of room temperature stability 
 

Protocols, Manuals & Usage

Protocols

  1. LunaScript Multiplex One-Step RT-PCR Kit Protocol (NEB #E1555)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. Can I set up my LunaScript Multiplex One-Step RT-PCR reactions at room temperature?
  2. Can I add random hexamers or oligo (dT) primers to LunaScript Multiplex One-Step RT-PCR reactions?
  3. Can I reduce the reaction volume in the LunaScript Multiplex One-Step RT-PCR reactions?
  4. How can I determine the annealing temperature for my LunaScript Multiplex One-Step RT-PCR reactions?
  5. Can I increase the reverse transcription time beyond the 10 minutes recommended in the LunaScript Multiplex One-Step RT-PCR protocol?
  6. Are the PCR products generated with the LunaScript Multiplex One-Step RT-PCR Kit compatible with a TA cloning system?
  7. What is the lowest RNA input amount recommended for the LunaScript Multiplex One-Step RT-PCR Kit?
  8. What can I do if a particular amplicon failed in a multiplex reaction using the LunaScript Multiplex One-Step RT-PCR Kit?
  9. Can I use the LunaScript Multiplex One-Step RT-PCR Kit to amplify long amplicons? 
  10. Can the LunaScript Multiplex One-Step RT-PCR Kit be used in the ARTIC workflow for RNA viral genome sequencing? 
  11. How do I choose between One-Step RT-PCR and Two-Step RT-PCR protocols?
  12. How do I choose between LunaScript Multiplex One-Step RT-PCR Kit and OneTaq One-Step RT-PCR Kit (NEB #E5315)?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

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]

Certificate Of Analysis

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]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

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

Notice to Purchaser: Nucleic acid-based aptamers for use with reverse transcriptases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 7,947,447; 8,404,830; 8,975,388 and their foreign equivalents). New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the WarmStart® Luna Reverse Transcriptase for Research Purposes Only. Commercial use of the WarmStart® Luna Reverse Transcriptase requires a license from New England Biolabs, Inc. Please contact [email protected] for more information.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

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