QIAcuity OneStep Advanced Probe Kit

• HotStart RT feature enabling simultaneous Nanoplate setup for QIAcuity Four and Eight
• Reaction setup at room temperature thanks to HotStart RT feature
• For singleplex and multiplex reactions
• 4x concentrated PCR Master Mix for loading more sample
• Advanced robustness against inhibitors

The QIAcuity OneStep Advanced Probe PCR Kit enables sensitive quantification of RNA or RNA+DNA targets on the QIAcuity digital PCR instrument in one combined RT-PCR reaction. It contains a 4x concentrated Master Mix and a 100x concentrated RT Mix optimized for microfluidic use in the QIAcuity Nanoplates. The kit enhances the specificity and efficiency of probe-based digital PCR to provide accurate, singleplex or up to 5-plex analysis.

The new kit is equipped with a HotStart Reverse Transcription (RT) Enzyme, enabling room-temperature reaction setup and simultaneous Nanoplate setup for loading into QIAcuity Four or Eight instruments.

The kit works in conjunction with the QIAcuity Digital PCR System and the QIAcuity Nanoplates.

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Advanced reverse transcription performance

The new QIAcuity Advanced Reverse Transcription Mix includes QIAGEN´s new patent-pending HotStart reverse transcription technology. The new HotStart RT enzyme is non-functional at room temperature and prevents the generation of off-target cDNA even when left to incubate at 37°C for many hours (see figure  Melt curve analysis of the new HotStart RT). It thereby enables a reaction setup at room temperature. The HotStart RT enzyme allows users to assemble their reactions at room temperature. Furthermore, and more importantly, the HotStart RT enzyme enables users to assemble and load multiple Nanoplates in parallel without needing to worry about unwanted and potentially unspecific RT activity while the plates wait at ambient temperatures. The HotStart RT enzyme is blocked while Nanoplates sit and wait inside the QIAcuity instrument until transferred to the thermal cycler, where the RT step of the protocol occurs.

Enhancer GC boosts specific assay performance

dPCR assay performance depends on the sequences and design of primer and probes. The GC content and amplicon length vary for industry catalog and custom-designed assays. PCR chemistries are optimized for universal usage and best performance; however, sometimes the addition of specific enhancers is recommended. The new Enhancer GC is an optional component (separate vial) you can add to the dPCR reaction when amplicons are GC-rich or are longer than 150 nt. It is also recommended for use with TaqMan (Thermo Fisher) gene expression assays.

Table: RT efficiency with and without Enhancer GC

Superior amplification performance

The QIAcuity OneStep Advanced Probe Kit for hydrolysis probe-based dPCR uses the latest version of QIAGEN's high-quality DNA Polymerase. The unique combination of QIAGEN's proprietary and well-proven buffer technology optimized for the Nanoplate microfluidic along with the new QuantiNova DNA Polymerase delivers highly consistent results in terms of sensitivity, reproducibility and efficiency.

Internal Control RNA

The QIAcuity OneStep Advanced Probe Kit comes with a QuantiNova Internal Control RNA (QN IC RNA), which can be used optionally as a reverse transcription and amplification control. The QuantiNova IC Probe Assay (200) (Cat. No. 205813), available for purchase separately, detects the QuantiNova Internal Control RNA over a 200 bp amplicon in the yellow channel on the QIAcuity instrument. The primer and probe sequences for the detection of the QuantiNova IC RNA have been bioinformatically validated for non-homology against hundreds of eukaryotic and prokaryotic organisms. Additionally, they have been experimentally tested against a multitude of human, mouse and rat RNA samples from multiple tissues and cell lines.

NOTE: Users should not purchase the QuantiNova IC Probe Assay Red 650 (500) (Cat. No. 205824), as it is not optimized to work with QIAcuity.

The QIAcuity OneStep Advanced Probe Kit contains a ready-to-use 4x PCR Master Mix, eliminating the need to optimize reaction and cycling conditions. The OneStep chemistry allows the RT and amplification for viral RNA of interest in a single step. In the OneStep reaction, RNA and DNA targets can also be detected together, for example, viral RNA with bacterial DNA.

The principle of the dPCR reaction in the nanoplates is described here.

The QIAcuity OneStep Advanced Probe Kit contains a ready-to-use 4x PCR master mix, eliminating the need to optimize reaction and cycling conditions. Simply add the 100x RT mix, template RNA and primer-probe sets to the master mix and follow the procedure outlined in the Quick-Start Protocol to get fast and reliable results on the QIAcuity. With the new HotStart RT enzyme, the parallel setup and simultaneous loading of multiple Nanoplates into QIAcuity Four and Eight instruments are possible (see figure  Nanoplate setup and loading).

The QIAcuity OneStep Advanced Probe Kit, combined with the QIAcuity Digital PCR System and the QIAcuity Nanoplates, enables quantitative analysis of RNA targets for various applications, including viral RNA or mRNA detection. Applications that require a simultaneous detection of RNA and DNA targets are also possible, for example, in wastewater testing.