Reaction Setup: For best results, we recommend running each RNA standard and sample in triplicate.

COMPONENT	20 µL REACTION	FINAL CONCENTRATION
Luna Universal Probe One-Step Reaction Mix (2X)	10 µL	1X
Luna WarmStart® RT Enzyme Mix (20X)	1 µL	1X
Forward primer (10 µM)	0.8 µL	0.4 µM
Reverse primer (10 µM)	0.8 µL	0.4 µM
Probe (10 µM)	0.4 µL	0.2 µM
Template RNA	variable	< 1 µg (total RNA)
Nuclease-free Water	to 20 µL

1. Thaw Luna Universal Probe One-Step Reaction Mix and other reaction components at room temperature, then place on ice. After thawing completely, briefly mix each component by inversion, pipetting or gentle vortexing.
2. Determine the total volume for the appropriate number of reactions, plus 10% overage and prepare assay mix of all components except RNA template accordingly. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation.
3. Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.
4. Add RNA template to qPCR tubes or plate. Seal tubes with flat, optically transparent caps; seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.
5. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).
6. Program real-time instrument with indicated thermocycling protocol (see table below). Ensure a plate read is included at the end of the extension step.