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 two different formats:
a.) LunaScript RT SuperMix Kit – first choice for qPCR (#E3010)
LunaScript RT SuperMix Kit is optimized for first strand cDNA synthesis in the context of a two-step RT-qPCR workflow. It 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.
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). This way you can benefit from maximum flexibility in cDNA synthesis. The LunaScript RT Master Mix Kit (Primer-free) is ideally suited for fast and reliable cDNA synthesis of molecules up to 9kb in length and therefore optimal as a universal cDNA synthesis kit for all your RT-PCRs, RT-qPCRs or RNA-seq experiments.
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.