Probe-based qPCR/RT-qPCR monitors an increase in fluorescence upon 5 → 3 exonuclease cleavage of a quenched, targetspecific probe to measure DNA amplification at each cycle of a PCR. At a point where the fluorescence signal is confidently detected over
the background fluorescence, a quantification cycle or Cq value can be determined. Cq values can be used to evaluate relative target abundance between two or more samples, or to calculate absolute target quantities in reference to an appropriate standard curve derived from a series of known dilutions.
In the Luna Universal One-Step Probe RT-qPCR Kit, Hot Start Taq DNA Polymerase is combined with a novel WarmStart-activated
reverse transcriptase, allowing dual control of enzyme activity via reversible, aptamer-based inhibition. This temperature-dependent activation helps to prevent undesirable non-specific priming and extension prior to thermocycling, providing added security for setting up reactions at room temperature. The engineered WarmStart Luna Reverse Transcriptase also possesses higher thermostability than many other RTs, allowing an optimal reaction temperature of 55°C. For difficult targets/templates, higher RT step temperatures of up to 60°C can be used without compromising Luna performance.
Note that to ensure full activation of the WarmStart Luna RT, incubation at temperatures lower than 50°C is not recommended.
The Luna Universal Probe One-Step Reaction Mix is supplied at 2X concentration and contains Hot-Start Taq DNA Polymerase, dNTPs,
and all required buffer components. It is formulated with a unique passive reference dye that is compatible across a variety of instrument platforms, including those that require a high or low ROX reference signal. The Reaction Mix also features dUTP for carryover prevention and a non-fluorescent visible dye for monitoring reaction setup. This visible dye does not overlap spectrally with fluorophores commonly used in qPCR and does not interfere with real-time detection.
The Luna WarmStart RT Enzyme Mix is supplied at 20X concentration and contains Luna WarmStart Reverse Transcriptase as well as
Murine RNase Inhibitor to aid in preventing RNA degradation (see also template preparation in Usage Notes). It is compatible with various RNA sample types (total RNA, poly(A)-RNA, etc.) and sources.