LunaScript® Multiplex One-Step RT-PCR Kit

Catalog #	Concentration	Size	List Price	Quantity	Your Price
E1555L		250 reactions	$1,466.00		$1,319.40
E1555S		50 reactions	$377.00		$339.30

Catalog #	Size	List Price	Your Price
E1555L	250 reactions	$1,466.00	$1,319.40
E1555S	50 reactions	$377.00	$339.30

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).

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.

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.

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.

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

E1555S -20

LunaScript® Multiplex One-Step RT-PCR Enzyme Mix	M1556SVIAL	-20	1 x 0.05 ml	25 X
LunaScript® Multiplex One-Step RT-PCR Reaction Mix	M1557SVIAL	-20	1 x 0.25 ml	5 X
Nuclease-free Water	B1502AVIAL	-20	1 x 1.5 ml

E1555L -20

LunaScript® Multiplex One-Step RT-PCR Enzyme Mix	M1556LVIAL	-20	1 x 0.25 ml	25 X
LunaScript® Multiplex One-Step RT-PCR Reaction Mix	M1557LVIAL	-20	1 x 1.25 ml	5 X
Nuclease-free Water	B1502AVIAL	-20	4 x 1.5 ml

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

An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

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FAQs

Quality Control Assay

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]

E1555S_L_v1

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]

Safety Data Sheets

Nuclease-free Water

Legal And Disclaimer

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 busdev@neb.com.

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.

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 busdev@neb.com 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.