INSTRUCTION MANUAL

LunaScript® RT SuperMix
NEB #M3010L/X/E 100/500/2,500 reactions
Version 1.0_4/22

Table of Contents
Introduction ..................................................................................................................................................................................................... 1
General Tips and Considerations ..................................................................................................................................................................... 1
Protocol ............................................................................................................................................................................................................ 2
Troubleshooting ............................................................................................................................................................................................... 3
Ordering Information ....................................................................................................................................................................................... 3
Revision History ..............................................................................................................................................................................................4

This product should be stored at –20°C, protected from light, and has a shelf-life of 2 years. LunaScript RT SuperMix usually remains
unfrozen at -20°C. The product is stable for at least 30 freeze/thaw cycles, and for short-term storage may be stored at 4°C for up to
1 month or at room temperature for up to 1 week.
This product is provided in volumes sufficient for preparation of up to 100 reactions (NEB #M3010L), 500 reactions (NEB #M3010X) or
2,500 reactions (NEB #M3010E).

Required Materials Not Included

RNA template
PCR strip tubes or microcentrifuge tubes
Thermocycler

Introduction
LunaScript RT SuperMix is an optimized master mix containing all the necessary components for first strand cDNA synthesis in the
context of 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. This product
offers robust, linear, and sensitive detection using total RNA inputs as high as 1 µg and as low as single copies of RNA, generating cDNA
products ideal for 2-step RT-qPCR detection.

General Tips and Considerations

Template RNA
• RNA of high purity enables the most sensitive RT-qPCR assays (1,2).

• Total RNA or mRNA can be used as input for first strand cDNA synthesis. Total RNA is generally sufficient for most RT-qPCR
experiments and can be prepared using typical column-based methods, e.g. Monarch® RNA Miniprep Kit (NEB #T2010). If desired,
mRNA can be easily purified using a polyA Spin™ mRNA Isolation Kit (NEB #S1560) or Magnetic mRNA Isolation Kit (NEB
#S1550).

• The amount of RNA required for detection depends on the abundance of the transcript-of-interest. In general, 1 ng to 1 μg total RNA or
0.1 ng to 100 ng mRNA are recommended.

1
cDNA Synthesis
• LunaScript RT SuperMix can perform cDNA synthesis in the temperature range of 45°C to 65°C. 55°C is optimal for most applications.

• In general, we recommend the transfer of 1 µl cDNA product into a 20 µl qPCR experiment. Where useful, up to 20% qPCR volume can
be undiluted cDNA product (e.g. 4 µl cDNA product into a 20 µl Luna qPCR reaction without significantly compromising
performance).

• The presence of genomic DNA or carry-over products can interfere with the accurate quantitation of target RNA, particularly for low
copy targets. No-RT control reactions can be simulated by heat-inactivation of the LunaScript RT SuperMix at 95°C for 1 minute before
adding RNA templates. In addition, no template control (NTC) reactions should be set up to demonstrate that positive reactions are
meaningful.

LunaScript RT SuperMix Protocol

1. Mix components briefly and spin down if necessary.

2. Prepare cDNA synthesis reaction as described below:

COMPONENT 20 µl REACTION FINAL CONCENTRATION

LunaScript RT SuperMix (5X) 4 µl 1X
RNA Sample variable (up to 1 µg)*
Nuclease-free Water to 20 µl

*Up to 1 µg total RNA, 1 µg mRNA or 100 ng specific RNA can be used in a 20 µl reaction.
However, the cDNA input for downstream qPCR detection should typically contain < 109 copies of the target
to ensure that quantitation remains linear. To accommodate larger amounts of input RNA (> 1 µg), the reaction
should be scaled up to ensure optimum cDNA synthesis.

For no template controls, mix the following components:

COMPONENT 20 µl REACTION FINAL CONCENTRATION

LunaScript RT SuperMix (5X) 4 µl 1X
Nuclease-free Water 16 µl

Incubate reactions in a thermocycler with the following steps:

CYCLE STEP TEMP TIME CYCLES

Primer Annealing 25°C 2 minutes
cDNA Synthesis 55°C 10 minutes 1
Heat Inactivation 95°C 1 minute

2
Troubleshooting Guide
Note: For additional assistance please refer to product FAQ's at www.neb.com/M3010.

PROBLEM POSSIBLE CAUSE(S) SOLUTION(S)

• Check the integrity of the RNA using denaturing agarose gel
electrophoresis (2) or BioAnalyzer® (Agilent Technologies)
• RNA should have a minimum A260/A280 ratio of 1.8 or higher.
Ethanol precipitation followed by a 70% ethanol wash can remove
Insufficient quality or quantity of most contaminants such as EDTA and guanidinium. Precipitation
Low cDNA yield
starting material with lithium chloride can remove polysaccharides (2).
• Phenol/chloroform extraction and ethanol extraction can remove
contaminant proteins such as proteases (2)
• Use sufficient amount of input RNA

References
1. Fleige, S. and Pfaffl (2006) Molecular Aspects of Medicine 27, 126–139.
2. Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual (3rd Ed.) Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.

Ordering Information
NEB # PRODUCT SIZE
M3010L/X/E LunaScript RT SuperMix 100/500/2,500
reactions

COMPANION PRODUCTS
NEB # PRODUCT SIZE
M3003S/L Luna Universal qPCR Master Mix 100/500 reactions
M3003X Luna Universal qPCR Master Mix 1,000 reactions
M3003E Luna Universal qPCR Master Mix 2,500 reactions
M3004S/L Luna Universal Probe qPCR Master Mix 200/500 reactions
M3004X Luna Universal Probe qPCR Master Mix 1,000 reactions
M3004E Luna Universal Probe qPCR Master Mix 2,500 reactions
T2010S Monarch RNA Miniprep Kit 50 preps
S1560S polyA Spin mRNA Isolation Kit 8 isolations
S1550S Magnetic mRNA Isolation Kit 25 isolations
B1500S/L Nuclease-free Water 25/100 ml

3
Revision History
REVISION # DESCRIPTION DATE
1.0 N/A 4/22

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.
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. The use of trademark symbols does not
necessarily indicate that the name is trademarked in the country where it is being read; rather, it indicates where the document was originally developed.
For more information about commercial rights, please email us at busdev@neb.com While NEB develops and validates its products for various applications, the use of this
product may require the buyer to obtain additional third party intellectual property rights for certain applications.
B CORPORATION® is a registered trademark of B Lab IP, LLC, Inc.
BIOANALYZER® is a registered trademark of Agilent Technologies, Inc.
© Copyright 2022 New England Biolabs, Inc.; all rights reserved

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