QIAcuity OneStepAdvanced Probe Kit (5mL)

QIAGEN offers the QIAcuity OneStep Advanced Probe Kit for use with the QIAcuity Digital PCR System and QIAcuity Nanoplates, enabling quantitative analysis of RNA targets in applications such as viral RNA or mRNA detection, as well as workflows that require simultaneous detection of RNA and DNA targets, including wastewater testing.

According to the manufacturer's quick-start protocol, the kit is intended for 1-step RT-dPCR and supports single-plex or multiplex reactions on 26K and 8.5K nanoplates. For reaction setup, the 4x OneStep Advanced Probe MM is used at 3 µL per reaction on the 8.5K nanoplate or 10 µL per reaction on the 26K nanoplate to reach a 1x final concentration, together with the 100x OneStep Advanced RT Mix at 0.12 µL (8.5K) or 0.4 µL (26K) for 1x reverse transcription activity, and a 20x primer-probe mix at 0.6 µL (8.5K) or 2 µL (26K). Total reaction volume is 12 µL for the 8.5K nanoplate and 40 µL for the 26K nanoplate. An optional Enhancer GC is recommended for Applied Biosystems TaqMan assays, amplicons longer than 150 bp, GC-rich amplicons, and RNA targets with challenging secondary structures.

The recommended thermal cycling program for the OneStep Advanced Probe MM comprises reverse transcription at 50°C for 40 min, RT enzyme inactivation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 5 s and combined annealing/extension at 58°C for 60 s. Reaction mixes can be assembled at room temperature thanks to the kit's hot-start capability, and a master mix volume 10% greater than the calculated total is recommended to cover controls.