LunaScript Multiplex One-Step RT-PCR Kit Protocol (NEB #E1555)
- Thaw the frozen components at room temperature. After thawing completely, mix the Reaction Mix thoroughly.*
- Briefly centrifuge all components to collect liquid to the bottom of the tubes, then place on ice.
- Prepare reactions as described below:
25 µl REACTION
LunaScript Multiplex One-Step RT-PCR Reaction Mix (5X)
LunaScript Multiplex One-Step RT-PCR Enzyme Mix (25X)
1–4 µM total**
up to 1 µg
to 25 µl
*Precipitates may be visible upon thawing. To ensure optimal performance, resuspend completely by pipetting up and down, inverting, or by vortexing then briefly centrifuging to collect all liquid to the bottom of the tube.
** The final primer concentration is typically 0.5 µM per primer (1 µM total) for single-plex RT-PCR. For multiplex RT- PCR, a total primer concentration of 1–2 µM (final) is recommended for reactions with up to 15 targets. For reactions with more targets, it may be necessary to empirically determine the appropriate primer concentration but typically, a total concentration of 1–4 µM (final) is recommended.
Optional: No-RT Negative Control Reaction
Prepare the no-RT reaction as in Step 3 above, leaving out the RNA sample. In a thermal cycler with a heated lid, heat the reaction at 98°C for 1 minute to inactivate the Luna RT, then let the reaction cool to 25°C. When cool, transfer the reaction to ice and add the RNA sample. Proceed to Step 4.
- Incubate the reactions in a thermal cycler with the following cycling conditions:
RT Inactivation/ Initial Denaturation
*A 55°C RT step temperature is optimal for Luna WarmStart Reverse Transcriptase. To ensure best performance and full WarmStart activation, avoid using an RT temperature below 50°C.
- The amplified cDNA product can be stored overnight at 4°C or at -20°C for long-term storage, before proceeding to downstream applications. 5–10 µl of the PCR product can be directly examined by agarose gel electrophoresis. The product can also be cloned using the NEB® PCR Cloning Kit (NEB #E1202). For downstream NGS library construction, a cleanup step with SPRI® beads is recommended, using the manufacturer’s stated protocol.
** Use of the NEB Tm Calculator is highly recommended as optimal annealing temperatures tend to be higher for Q5 Hot Start High-Fidelity DNA Polymerase than for other polymerases. Alternative online calculators may underestimate the optimal annealing temperature.
*** An annealing time of 10–20 seconds is recommended for single-plex reactions; it should be increased when performing multiplex RT-PCR. For example, a 1 minute annealing time is generally sufficient for a 10-plex RT-PCR assay. When the final concentration of each primer is less than 0.05 µM, longer extension time may be needed.