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. The LunaScript RT SuperMix contains random hexamer and poly-dT primers, allowing even coverage across the length of the RNA targets. In addition, the LunaScript RT SuperMix contains a blue dye, providing a visual indicator that can be followed throughout the two-step RT-qPCR process. The LunaScript RT SuperMix offers robust, linear, and sensitive detection using total RNA inputs as high as 1 µg and as low as single copies of RNA.
Comparison of one-step and two-step RT-qPCR workflows
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 (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.
The LunaScript RT SuperMix Kit 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 the 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.
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 (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.
At just 13 minutes, the LunaScript RT SuperMix Kit offers the shortest available first-strand cDNA synthesis protocol Comparison of recommended protocols for cDNA synthesis. The LunaScript RT SuperMix Kit requires the shortest reaction time and tolerates elevated temperatures, reducing complications from RNA secondary structure.
Kit Components
The following reagents are supplied with this product:
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