Product Class: Kit

LunaScript® RT Master Mix Kit (Primer-free)


Catalog #E3025

Product Introduction

The LunaScript RT Master Mix Kit (Primer-free) features an optimized 5X master mix containing all the necessary components for first strand cDNA synthesis, except primers.

  • 5X master mix contains Luna® Reverse Transcriptase, dNTPs, and Murine RNase Inhibitor
  • Primer-free format enables user flexibility for choice of primers for optimal cDNA synthesis
  • Complete first strand cDNA synthesis protocol in less than 15 minutes
  • Can be used for first strand cDNA synthesis, two-step RT-qPCR, two-step RT-PCR, and RNA-seq workflows
  • Non-interfering, visible tracking dye helps to eliminate pipetting errors
  • Includes No-RT control mix for increased confidence
 

Product Information

Description

The LunaScript RT Master Mix Kit (Primer-free) includes an optimized 5X master mix containing all the necessary components for first strand cDNA synthesis, except primers. The mix features the thermostable Luna Reverse Transcriptase, which supports cDNA synthesis at elevated temperatures. Murine RNase Inhibitor is included to protect template RNA from degradation, along with dNTPs. In addition, the presence of a blue tracking dye provides a visual indicator for the RT step and downstream applications.

The mix is compatible with random primers, oligo dT primers, and gene-specific primers, enabling maximum cDNA synthesis flexibility. The cDNA product generated by the LunaScript RT Master Mix (Primer-free) can be used in a variety of downstream applications. For real-time PCR quantitation, a mixture of random and dT primers is recommended to generate cDNA. The mix offers robust, linear and sensitive detection from 1 μg total RNA input down to low copies of RNA, similar to the LunaScript RT SuperMix Kit (NEB #E3010). To generate long or full-length cDNAs, oligo dT primers should be used. With a short incubation at 55°C for 10 minutes, the LunaScript RT Master Mix (Primer-free) can make cDNA products up to 9 kb. A No-RT control mix is provided for ease of setting up control reactions.

Figure 1: Comparison of supermix, master mix and cDNA synthesis kit formats





Figure 2: At just 13 minutes, LunaScript offers the shortest available first-strand cDNA synthesis protocol



Comparison of recommended protocols for cDNA synthesis. Both LunaScript RT SuperMix Kit and LunaScript RT Master Mix Kit require the shortest reaction time and tolerate elevated temperatures, reducing complications from RNA secondary structure.





Figure 3: LunaScript RT Master Mix (Primer-free) supports numerous applications



By adding different primers including a Random Primer Mix, d(T))23VN oligos, or random hexamers, the LunaScript RT Master Mix can produce cDNA that is ideally suited for downstream applications such as RT-qPCR, RT-PCR, and RNA-seq studies.





Figure 4: LunaScript RT Master Mix (Primer-free) offers robust 2-step RT-qPCR quantitation



A. In the presence of Random Primer Mix (NEB #S1330), the LunaScript RT Master Mix (Primer-free) can deliver linear cDNA yields across RNA inputs from 1 µg to 100 pg with linearity similar to LunaScript (NEB #E3010) in a 2-step RT-qPCR workflow.

B. For average transcripts, the use of d(T)23VN primers offers cDNA coverage similar to the Random Primer Mix. However, for long transcripts (e.g., SMG1), random primers often enable better coverage of the 5´ end of the target, as seen in the example in the lower righthand box where the RT-qPCR target was located at the 5´ end of the 16 kb SMG1 transcript; its detection by an anchored dT primer alone [d(T)23VN] gave delayed Cq values when compared to the performance using the Random Primer Mix (a mixture of random hexamers and anchored dT primer).





Figure 5: Better performance of LunaScript RT Master Mix (Primer-free) in 2-step RT-qPCR quantitation



A. Several first strand cDNA synthesis kits were used according to manufacturer’s recommendations to generate cDNA using the random primers provided with each product. Input included human total RNA from 1 µg – 100 pg. cDNA products were then evaluated by qPCR using eight targets varying in abundance, length and %GC. qPCR detection of the cDNA products was performed using the 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). More details on this visualization method can be found in the application note “Development of a high-throughput data analysis method for quantitative real-time PCR (qPCR).”

B. Results from four targets above (two typical control genes and two others) were plotted to examine Cq as a function of input. LunaScript gave the earliest Cq values when compared to other commercial kits.





Figure 6: LunaScript RT Master Mix (Primer-free) can generate a broad range of cDNA products for use in routine or high fidelity PCR



LunaScript RT Master Mix (Primer-free) was used to synthesize cDNA from 1 µg of Jurkat total RNA in the presence of d(T))23VN primers under the standard reaction condition of 55°C for 10 min, 95°C for 1 min. Subsequent to cDNA synthesis, different PCR amplification reagents can be used for PCR detection. For routine application up to 5 kb, OneTaq 2X Master Mix (NEB #M0482) is recommended. For highest fidelity, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) is recommended. For highest yield of long PCR amplification, LongAmp Taq 2X Master Mix (NEB #M0287) can be used (data not shown).
 
This product is related to the following categories:
Luna® qPCR & RT-qPCR Products,
RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products,
This product can be used in the following applications:
RT-PCR,
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),
RT-PCR & cDNA Synthesis

Properties & Usage

Features

  • Primer-free mix enables user flexibility for choice of primers for optimal cDNA synthesis
  • Complete first strand cDNA synthesis protocol in less than 15 minutes
  • Includes No-RT control mix for increased confidence
 

Protocols, Manuals & Usage

Protocols

  1. LunaScript RT Master Mix Kit Protocols (NEB #E3025)

Manuals

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

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

Troubleshooting

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 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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