QIAcuity OneStep Advanced EG Kit

For dye-based one-step RT-PCR on the QIAcuity digital PCR instruments

S_1344_9_LS_dPCR_QIAcuity_OneStep_Advanced_EG_Kit

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QIAcuity OneStep Advanced EG Kit (1ml)

Cat. No. / ID:  250141

OneStep Advanced EG Master Mix (4x), OneStep RT Mix (100x), 20 µl QN Internal Control RNA Q-Solution, RNase-free water; for 100 reactions in Nanoplate 26k and 333 reactions in Nanoplate 8.5k
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$540.00
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Kit
QIAcuity OneStep Advanced EG Kit (1 ml)
QIAcuity OneStep Advanced EG Kit (5 ml)
The QIAcuity OneStep Advanced EG Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

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Features

  • For dye-based RT-digital PCR reactions using EvaGreen
  • HotStart RT feature enabling simultaneous Nanoplate setup for QIAcuity Four and Eight
  • Reaction setup at room temperature thanks to HotStart RT feature
  • 4x concentrated PCR Master Mix for loading more sample

 

Product Details

The QIAcuity OneStep Advanced EG PCR Kit enables sensitive quantification of RNA or RNA+DNA targets in one combined RT-PCR reaction on the QIAcuity Digital PCR System. The kit contains a 4x concentrated Master Mix and a 100x concentrated RT Mix optimized for microfluidic use in the QIAcuity Nanoplates. The kit improves the specificity and efficiency of dye-based digital PCR to provide accurate quantification analysis. As an intercalating dye, EvaGreen binds to double-stranded DNA and enhances the quantitative accuracy of gDNA or cDNA measurements on the QIAcuity dPCR instruments.

The new kit is equipped with a HotStart Reverse Transcription (RT) Enzyme. Thanks to the RT Enzyme, you can work at room temperature to simultaneously set up your reaction and nanoplate for loading into QIAcuity Four or Eight instruments.

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

Advanced reverse transcription performance

The new QIAcuity Advanced Reverse Transcription Mix includes a 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. With this enzyme, you can set up your reactions  at room temperature. Importantly, with the HotStart RT enzyme you can assemble and load multiple nanoplates in parallel, at ambient temperatures, without risking  unwanted and potentially unspecific RT activity. The HotStart RT enzyme is blocked while nanoplates remain inside the QIAcuity instrument before transfer to the thermal cycler, where the RT step of the protocol occurs.

Dye-based detection with EvaGreen

The special Master Mix in the QIAcuity OneStep EG PCR Kit enables accurate double-stranded DNA target amplification and quantification. The kit includes an optimized reference dye needed for counting analyzable partitions in Nanoplates and dPCR analysis. Moreover, EvaGreen provides a higher fluorescence signal than SYBR Green at the same concentrations and delivers maximum amplification efficiency, specificity and sensitivity in dPCR.

 

Principle

The QIAcuity OneStep Advanced EG Kit contains a ready-to-use 4x PCR Master Mix, eliminating the need to optimize reaction and cycling conditions. With the OneStep chemistry you can perform 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.

 

Procedure

The QIAcuity OneStep Advanced EG Kit contains a ready-to-use 4x PCR Master Mix, so you don’t 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 Digital PCR System. 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).

See figures

Applications

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.

 

Supporting data and figures

Resources

Kit Handbooks (1)
Safety Data Sheets (1)
Certificates of Analysis (1)

FAQ

Is this kit approved for diagnostic use?
No. This kit is for research use only.
FAQ ID - 4012
What is the lower limit of target that can be quantified in a reaction using the QIAcuity One-Step Advanced EvaGreen kit?
Ten copies of a target can be quantified in a single reaction on a 8.5k and 26k Nanoplate.
FAQ ID - 4014
Can I use this kit to quantify RNA and DNA together?
Yes. You can quantify RNA and DNA molecules with the OneStep Advanced EvaGreen Kit.
FAQ ID - 4011
Do QNIC assays come with the kit?
No.
FAQ ID - 4013
Can I reduce reaction volumes?
No. It's critical to use 12 µL reaction volumes for the 8.5k Nanoplates and 40 µL reaction volumes for the 26k Nanoplates. Using lower reaction volumes results in priming errors, which lead to errors in quantification.
FAQ ID - 4015
I have a single-plate instrument and would like to run multiple plates back-to-back. Since there is a HotStart RT in the new kit, can I assemble multiple plates at once and store them at room temperature while the other plates are running?
No. While the HotStart RT would prevent premature RT, the plates would be left unprimed for hours. This would result in undesirable pre-priming of the plates over time, which would lead to overpriming of the Nanoplate wells.
FAQ ID - 4016