Robust cDNA-synthesis for RT-qPCR and endpoint RT-PCR
Two-step RT-qPCR uncouples cDNA synthesis and subsequent qPCR, allowing greater freedom in selecting reverse transcriptases (RTs) and qPCR reagents separately. This flexibility can be useful for controlling sequence representation, efficiency, and when working with difficult RT-qPCR reactions or low RNA inputs.
LunaScript reverse transcriptase: faster, hotter and more reliable!
LunaScript reverse transcriptase is a unique, novel in silico designer enzyme that works particularly fast and reliably on almost all RNAs and input amounts and at elevated temperatures, resolving even “difficult” secondary structures of RNA.
- Simple reaction setup due to convenient SuperMix/Master Mix format
- Unique in silico designer enzyme
- Available with primers (#E3010) or without primers (#E3025) in the mix – for maximum flexibility
- Synthesize cDNA in less than 15 minutes
- Easier analysis of RNAs with secondary structures thanks to the increased reaction temperature of 55°C (up to 65°C)
- Incl. No-RT control – for even more safety against non-specific reactions
- Eliminate pipetting errors with non-interfering, visible tracking dye
- Enjoy consistent linearity, sensitivity, and capacity for reliable RNA quantification
LunaScript offers the fastest 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.
LunaScript reverse transcriptase is available in three different formats:
a.) LunaScript RT SuperMix Kit – first choice for qPCR (#E3010 –including negative control, #M3010 – only SuperMix)
LunaScript RT SuperMix Kit is optimized for first strand cDNA synthesis in the context of a two-step RT-qPCR workflow. It contains an optimizes buffer, dNTPs, RNase inhibitor as well as random-hexamer and oligo-d(T) primers. You simply add your RNA template.
In the convenient kit format (#E3010) the LunaScript RT SuperMix (#M3010) is delivered including a vial of nuclease-free and a primer set used as “no-RT-control”.
Luna delivers best-in-class performance, user-friendly protocols, and includes a convenient blue dye to track your sample.
The cDNA products generated by LunaScript have been extensively evaluated in qPCR using the Luna qPCR Master Mixes. In combination, these products provide a two-step RT-qPCR workflow with excellent sensitivity and accurate, linear quantitation.
Applications:
- Production of high-quality cDNAs for subsequent qPCR
- Production of cDNAs for ONT SARS-CoV-2 Seq
b.) LunaScript RT Master Mix Kit (Primer-free) (#E3025)
The best-in-class LunaScript enzyme is also available in a primer-free master mix (#E3025). You can either use random-hexamer or oligo-d(T) primers or gene-specific primers. This way you can benefit from maximum flexibility in cDNA synthesis.
Applications:
- Ideally suited for fast and reliable cDNA synthesis of molecules up to 9kb in length
Universal cDNA synthesis kit for all your RT-PCRs, RT-qPCRs or RNA-seq experiments
c.) LunaScript Multiplex One-Step RT-PCR Kit (#E1555) – the “high-fidelity” kit
The LunaScript Multiplex One-Step RT-PCR Kit offers an optimized protocol for cDNA-synthesis and amplification in one reaction. Dual temperature control of enzyme activities by aptamer technology allows to set up reactions at room temperature and store them at room temperature for up to 24h. Due to the 25x concentration of the enzyme mix, the kit allows to perform RT-PCRs up tp 50-plex with amplicons at 3kb length. In addition to the RNA-template, simply add the desired RT and PCR primers and obtain highly specific, “error-free” amplicons.
Applications:
- Suitable for sensitive and accurate one-step RT-PCR experiments with fragments up to 3 kb length
- Both single-plex and up to over 50-plex amplifications are possible
The LunaScript RT SuperMix Kit offers exceptional sensitivity, linearity, and reproducibility in two-step RT-qPCR workflows
RNA was converted to cDNA using the 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 the Luna Universal qPCR Master Mix (#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 5×10^9 to 50 copies of ERCC00130 (~10 ng – 10 fg) was converted to cDNA and then quantitated by qPCR.
The LunaScript RT SuperMix Kit 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 the Luna Universal qPCR Master Mix (#M3003) or Luna Universal Probe qPCR Master Mix (#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).
LunaScript RT Master Mix (Primer-free) – the “universal” cDNA-synthesis kit for 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.
Product Table
| PRODUCT | Prod.Nr. | SIZE | INFO | |
|---|---|---|---|---|
| LunaScript™ RT SuperMix | M3010L | 100 rxns |  |
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| M3010X | 500 rxns | |
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| M3010E | 2500 rxns | |
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| LunaScript™ RT SuperMix Kit | E3010S | 25 rxns | |
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| E3010L | 100 rxns | |
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| LunaScript RT Master Mix Kit (Primer-free) | E3025S | 25 rxns | |
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| E3025L | 100 rxns | |
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| Luna® Universal One-Step RT-qPCR Kit | E3005S | 200 rxns | |
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| E3005L | 500 rxns | |
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| E3005X | 1000 rxns | |
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| E3005E | 2500 rxns | |
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| Luna® Universal Probe One-Step RT-qPCR Kit | E3006S | 200 rxns | |
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| E3006L | 500 rxns | |
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| E3006X | 1000 rxns | |
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| E3006E | 2500 rxns | |
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| LunaScript Multiplex One-Step RT-PCR Kit | E1555S | 50 rxns | |
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| E1555L | 250 rxns | |
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As of 01.01.2024
Further information can be found in our Technical Resources section or at neb.com. Information on trademarks can be found here.
