Built-in visual indicator for accurate reaction setup
The master mix supplied with QuantiNova RT-PCR Kits 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 template nucleic acid is diluted in QuantiNova Yellow Template Dilution Buffer and added to the master mix, the color of the solution changes from blue to green (see figure “ Accurate reaction setup indicated by the built-in pipetting control”), providing a visual indication that each reaction was set up correctly.
QuantiNova SYBR Green and QuantiNova Probe RT-PCR Kits
QuantiNova SYBR Green and QuantiNova Probe RT-PCR Kits use 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 (SYBR Green and multiplex kits) or 45°C (probe kit) 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 (SYBR Green and multiplex kits) or 45°C (probe kit), 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 (SYBR Green and multiplex kits) or 5 minutes (probe kit) 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 (see figures “Consistent results after room temperature storage” and “ Convenient room-temperature reaction setup without compromising performance”), preventing the creation of artifacts and also facilitating automated reaction setup.
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 (see figure “Accurate quantitation with very high or very low input amounts”).
The gDNA reduction step in QuantiNova Probe RT-PCR procedures minimizes misquantification caused by genomic DNA carryover. Reduction of genomic DNA is crucial for accurate gene expression results and eliminates the need to design RNA-specific primers or probes. With integrated gDNA reduction (see figure “ Increased reliability of gene expression results using gDNA reduction”), time is saved and costs are reduced, since a separate DNase digestion during or after purification of RNA samples is not necessary.
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 (see figure “Reliable monitoring of RT-PCR inhibition”), it can be used to confirm successful reverse transcription and amplification. Duplex capability also enables inclusion of the internal control or a reference gene for direct comparison with the target gene.
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.
QuantiNova Multiplex RT-PCR Kits
The specialized master mix supplied with the QuantiNova Multiplex RT-PCR Kit allows easy setup of multiplex reactions and ensures that results are comparable to singleplex RT-PCR. The highly concentrated 4x master mix accommodates up to 800 ng template input and offers outstanding sensitivity even with up to 5plex reactions.
The kit can clearly distinguish between small differences in the amount of template and provide accurate quantification of targets of widely differing abundance (see figure “Highly sensitive multiplex detection of targets with varying abundance”). Additionally, a simple protocol for direct amplification from cultured cells is provided, enabling RNA analysis even from a single cell (see figure “Multiplex gene expression analysis in single cells.”). The unique two-phase hot-start procedure (see figure “Principle of the novel QuantiNova two-phase hot-start mechanism”) ensures outstanding specificity and allows room-temperature reaction setup, which is conveniently suited for automated procedures. The specially developed PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times, allowing multiplex qRT-PCR in about one hour. The addition of a visual pipetting control increases in-process safety by minimizing human errors (see figure “Accurate reaction setup indicated by the built-in pipetting control”). The optional Internal Control RNA, which can be co-detected with targets, removes variation caused by inhibitors or other factors, allowing accuracy and reproducibility to be monitored. Overall, our ultrafast and in-process controlled multiplex qRT-PCR workflow increases efficiency by generating more insight from limited sample material.