For ultrafast, multiplex, real-time PCR and two-step qRT-PCR using sequence-specific probes

Performance

The special master mix supplied with QuantiNova Multiplex PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex PCR data that are comparable with singleplex PCR data. The highly concentrated 4x master mix accommodates up to 800ng template input ensuring outstanding sensitivity, even in up to 5-plex reactions. The kit can clearly distinguish between small differences in the amount of template and provides accurate quantification of targets of widely differing abundance.

A novel, antibody-mediated, hot-start mechanism (see figure Novel antibody mediated hot-start mechanism) ensures outstanding specificity and allows reaction set up at room temperature, ideally suited for automated procedures. The specially developed fast PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times, allowing multiplex qPCR in less than one hour. The visual pipetting control prevents human errors (see figure Built-in pipetting control) and increases process safety, particularly if combined with the Internal Control RNA provided in the QuantiNova Reverse Transcription Kit for quantitative 2-step RT-PCR.
The QuantiNova Multiplex PCR Kit increases workflow efficiency by generating more insight from limited sample material by using ultrafast and in-process controlled multiplex qPCR.

Principle

QuantiNova Multiplex PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization. The specially developed fast PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times (see figure "Fast primer annealing").

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. Additionally the Internal Control RNA provided in the QuantiNova Reverse Transcription Kit for quantitative 2-step RT-PCR can be incorporated to monitor successful reverse transcription and qPCR.
QuantiNova Multiplex PCR Buffer includes a balanced combination of K⁺ and NH₄⁺ ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer").

The master mix supplied with the QuantiNova Multiplex PCR Kit contains an inert blue dye that does not interfere with the real-time PCR, but increases visibility in the tube or well. The QuantiNova Yellow Template Dilution Buffer contains an inert yellow dye. When the template nucleic acid, diluted with the QuantiNova Yellow Template Dilution Buffer, is added to the master mix, the color of the solution changes from blue to green (see figure Built-in pipetting control), providing a visual indication of correct pipetting and reaction setup.

The QuantiNova Multiplex PCR Kit master mix can conveniently be stored at 2-8° C for up to 12 months, and also the reaction set up is extraordinary stable at room temperature, allowing automated procedures to increase efficiency and accuracy.

Procedure

QuantiNova Multiplex PCR Kits contain ready-to-use 4x master mix that eliminate the need for optimization of reaction and cycling conditions. Simply add up to 800ng template DNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits provide ROX passive reference dye in a separate tube, to adjust appropriate ROX concentration, if required for your instrument.

For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiNova Reverse Transcription Kit, which provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination. It additionally provides the QuantiNova Internal Control RNA, offering an in-process monitoring of successful reverse transcription and qPCR. 

Applications

QuantiNova Multiplex PCR Kits can be used for multiplex gene expression analysis of cDNA or gDNA targets on any real-time cycler. To fully exploit the capability of multiplex benefits, we recommend instruments providing up to 5-plex capacity, such as the Rotor-Gene Q.