The QuantiNova SYBR Green RT-PCR Kit uses a two-phase hot-start procedure (see figure
Principle of the novel QuantiNova two-phase hot-start mechanism). This includes heat-mediated activation of both the HotStaRT-Script reverse transcriptase at 50°C and the PCR polymerase at 95°C. At low temperatures the HotStaRT-Script forms a complex with an RT-Blocker molecule, leading to inactivation. At 50°C, the complex dissociates and the active HotStaRT-Script enzyme is released. The QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and a novel additive, QuantiNova Guard, which stabilizes the complex. This improves the stringency of the hot-start procedure and prevents extension of non-specifically annealed primers and primer–dimers. Within 2 minutes of raising the temperature to 95°C, the QuantiNova Antibody and QuantiNova Guard are denatured and the QuantiNova DNA Polymerase is activated, enabling PCR amplification. This unique two-phase hot-start enables reaction setup to remain stable for at least 2 hours at room temperature, preventing the creation of artifacts and also facilitating automated reaction setup.
The newly developed internal control is a defined transcript (RNA molecule) that can simply be added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup or the presence of inhibitors. Since the internal control behaves comparably to real transcripts, it can be used to confirm successful reverse transcription and amplification.
The master mix supplied with the QuantiNova SYBR Green RT-PCR Kit contains an inert blue dye that does not interfere with the reverse transcription or 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, providing a visual indication of correct pipetting and reaction setup (see figure
Accurate reaction setup indicated by the built-in pipetting control).
The QuantiNova DNA Polymerase and the unique composition of the RT-PCR buffer provide sensitive quantification of low-copy RNA targets, as well as accurate quantification over a wide linear range on any common cycler.