Protocol for Two-step RT-qPCR using the LunaScript^® RT SuperMix Kit (NEB #E3010) and the Luna^® Universal qPCR Master Mix (NEB #M3003) or Luna Universal Probe qPCR Master Mix (NEB #M3004) 

Step 1: First strand cDNA synthesis

1. Mix components briefly and spin down if necessary.
Note: LunaScript RT SuperMix and No-RT Control Mix are usually not frozen at -20°C.

2. Prepare cDNA synthesis reaction as described below.

*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.

3. Incubate reactions in a thermocycler with the following steps:

Step 2: qPCR reaction

The cDNA product can be directly used in qPCR reaction. In general, 1 μl cDNA product is recommended for usage in a 20 μl qPCR detection. When necessary, up to 20% qPCR volume can be undiluted cDNA product (e.g., 4 μl cDNA product in a 20 μl qPCR reaction).

We recommend using the Luna® Universal qPCR Master Mix (NEB #M3003) and Luna Universal Probe qPCR Master Mix (NEB #M3004) for qPCR detection, as cDNA products generated using the LunaScript RT SuperMix Kit have been extensively evaluated using the two Luna kits.

• Prepare dye-based qPCR detection as follows:

• Reaction setup for probe-based qPCR detection