Product Class: Kit

LunaScript® RT SuperMix Kit

LunaScript RT SuperMix Kit (NEB #E3010) includes supermix, No-RT control mix, and nuclease-free water.

Supermix only option now available, please see LunaScript RT SuperMix (NEB #M3010).

Featured in the ARTIC SARS-CoV-2 sequencing workflow (1) and a component in the NEBNext® ARTIC SARS-CoV-2 kits.

Product Introduction

  • Single-tube supermix contains random hexamer and oligo-dT primers, dNTPs, Murine RNase Inhibitor, and Luna® Reverse Transcriptase
  • Less than 15 minute first-strand cDNA synthesis protocol
  • Novel, thermostable Luna Reverse Transcriptase improves performance 
  • Combine with Luna qPCR master mixes (NEB #M3003, M3004) for robust RT-qPCR results
  • Eliminate pipetting errors with non-interfering, visible tracking dye
  • Primer-free version (NEB #E3025) available
  • Does include nuclease-free water and No-RT Control Mix

1. Josh Quick 2020. nCoV-2019 sequencing protocol v3 (LoCost). protocols.io 

Catalog # Size Concentration
E3010S 25.0 reactions
E3010L 100.0 reactions

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Product Information

Description

LunaScript RT SuperMix is an optimized master mix for first strand cDNA synthesis and can be used in amplicon sequencing or a two-step RT-qPCR workflow. It features the thermostable Luna Reverse Transcriptase, which supports cDNA synthesis at elevated temperatures. Murine RNase Inhibitor is also included to protect template RNA from degradation. LunaScript RT SuperMix contains random hexamer and poly-dT primers, allowing even coverage across the length of the RNA targets. In addition, LunaScript RT SuperMix contains a blue dye, providing a visual indicator that can be followed throughout the two-step RT-qPCR process. The product offers robust, linear, and sensitive detection using total RNA inputs as high as 1 µg and as low as single copies of RNA.

For RT-PCR, RNA-seq, and other applications, LunaScript RT Master Mix Kit (Primer-free) (NEB #E3025) is an optimized master mix containing all the necessary components for first strand cDNA synthesis, except for primers. The mix is compatible with random primers, oligo dT primers, and gene-specific primers, enabling maximum cDNA synthesis flexibility. Learn more here.

In comparison to the LunaScript kit version (NEB #E3010), this LunaScript product does not contain a No-RT Control Mix or nuclease-free water. More details can be found below:

Figure 1: Comparison of LunaScript Products





Figure 2: Product options for one-step and two-step RT-qPCR workflows





Figure 3: LunaScript RT SuperMix offers exceptional sensitivity, linearity and reproducibility in two-step RT-qPCR workflows



RNA was converted to cDNA using 1X LunaScript RT SuperMix in 20 μl reactions using standard reaction conditions (25°C/2 min, 55°C/10 min, 95°C/1 min). cDNA was then quantitated by qPCR using Luna Universal qPCR Master Mix (NEB #M3003) and 1 μl of cDNA product as template, with triplicate reactions at each input concentration.

 

A. A serial dilution of Jurkat total RNA (1 μg – 1 pg) was converted to cDNA and then quantitated by qPCR using a β-actin target.

 

B. ERCC (External RNA Controls Consortium) mix1 RNA containing 5x109 to 50 copies of ERCC00130 (~10 ng – 10 fg) was converted to cDNA and then quantitated by qPCR.




Figure 4: LunaScript RT SuperMix supports even coverage of long transcripts



Eight pairs of primers were designed to cover the entire 15.2 kb HERC1 transcript. Jurkat total RNA (1 μg – 1 ng) was converted to cDNA using 1X LunaScript RT SuperMix in 20 μl reactions using standard reaction conditions (25°C/2 min, 55°C/10 min, 95°C/1 min). cDNA coverage was then evaluated by qPCR using Luna Universal qPCR Master Mix (NEB #M3003) and 1 μl of cDNA product as template, with duplicate reactions for each target at each input concentration.




Figure 5: LunaScript RT SuperMix demonstrates superior linear detection of RNA targets



Commercially available cDNA supermixes were used according to manufacturer’s recommendations to generate cDNA from 1 μg – 100 pg human (Jurkat) total RNA. cDNA products were then evaluated by qPCR using eight targets varying in abundance, length and %GC. qPCR detection was performed using Luna Universal qPCR Master Mix (NEB #M3003) or Luna Universal Probe qPCR Master Mix (NEB #M3004). Results were evaluated for efficiency and ΔCq, where ΔCq measures low input detection and lack of non-template control (NTC) amplification (ΔCq = average Cq of NTC - average Cq of lowest input). Green box indicates target performance criteria (Efficiency = 90-110%, ΔCq ≥ 3).

 

Learn more about this “Dots in Boxes” visualization method.




Figure 6: At just 13 minutes, LunaScript RT SuperMix offers the shortest available first-strand cDNA synthesis protocol 



Comparison of recommended protocols for cDNA synthesis. LunaScript RT SuperMix requires the shortest reaction time and tolerates elevated temperatures, reducing complications from RNA secondary structure.




Figure 7: LunaScript RT SuperMix exhibits exceptional stability at room temperature



LunaScript RT SuperMix and other commercial products were left at 25°C for 15 days, and then used according to manufacturer’s recommendations to generate cDNA from 1 μg – 100 pg human (Jurkat) total RNA. cDNA products were then evaluated by qPCR using eight targets varying in abundance, length and %GC. qPCR detection was performed using Luna Universal qPCR Master Mix (NEB #M3003). Results were evaluated for efficiency and ΔCq, where ΔCq measures low input detection and lack of no-template control (NTC) amplification (ΔCq = average Cq of NTC - average Cq of lowest input). Green box indicates target performance criteria (Efficiency = 90-110%, ΔCq ≥ 3).

 

Learn more about this “Dots in Boxes” visualization method.




This product is related to the following categories:
Luna® qPCR & RT-qPCR Products
This product can be used in the following applications:
qPCR & RT-qPCR,
Reverse Transcription (cDNA Synthesis),
RNA Cloning

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Protocol for LunaScript®  RT SuperMix Kit (E3010)
  2. Protocol for Two-step RT-qPCR using the LunaScript® RT SuperMix Kit (NEB #E3010) and the Luna® Universal qPCR Master Mix (NEB #M3003) or Luna Universal Probe qPCR Master Mix (NEB #M3004) 

Manuals

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

Application Notes

FAQs & Troubleshooting

FAQs

  1. Can I use  RNA samples purified from different storage conditions such as RNALater in my LunaScript®  cDNA synthesis reactions?
  2. Can I add gene-specific primers in my LunaScript®  cDNA synthesis reaction?
  3. Can I set up my cDNA synthesis reaction at room temperature when using the LunaScript®  RT SuperMix?
  4. Can I use a shorter incubation time for my cDNA synthesis reaction when using the LunaScript®  RT SuperMix?
  5. Can I use LunaScript® cDNA products directly in qPCR? How much can I use for detection in a qPCR reaction?
  6. How much RNA template should I use in my LunaScript®  cDNA synthesis reactions?
  7. How should I store LunaScript®  cDNA products?
  8. How should I choose between a one-step RT-qPCR or two-step RT-qPCR approach?
  9. Should I include a No-Reverse Transcriptase (RT) control reaction when setting up my cDNA synthesis reaction?
  10. What qPCR reagents do you recommend for detection of cDNA products?
  11. What temperature should I use for my LunaScript®  cDNA synthesis reaction?
  12. Why do I have low cDNA yields?
  13. Will the blue dye in the LunaScript®  RT SuperMix interfere with detection?
  14. How do I choose between the first strand cDNA synthesis kits from NEB?
  15. When using LunaScript cDNA products in downstream PCR (i.e., two-step RT-PCR), what volumes and PCR products can be used? 
  16. Can I use LunaScript RT SuperMix in RNA-seq workflows?
  17. How do I choose between LunaScript® RT SuperMix Kit and ProtoScript first strand cDNA synthesis kits?
  18. Can I use a shorter incubation time for my cDNA synthesis reaction when using the LunaScript® RT Master Mix (Primer-free)? Does longer incubation increase the yield of cDNA?
  19. How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products?
  20. How do I know whether my template RNA is of good quality?

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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 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.

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