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.
The latest addition to the Luna qPCR/RT-qPCR portfolio, the 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 quantitations
- Simplify reaction setup with convenient supermix format
- Eliminate pipetting errors with non-interfering, visible tracking dye
- Synthesize cDNA in less than 15 minutes
- Experience best-in-class performance, as all Luna products have undergone rigorous testing to optimize specificity, sensitivity, accuracy and reproducibility
- Enjoy consistent linearity, sensitivity, and capacity for reliable RNA quantification
Combine LunaScript with Luna qPCR Master Mixes for robust RT-qPCR.
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.
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 5×109 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 (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).