QuantiNova RT-PCR Kits

• Unique two-phase hot-start procedure for room-temperature setup
• Visual pipetting control, resulting in fewer pipetting errors
• Internal control for positive in-process verification of successful RT-PCR
• Accurate quantification over several logs of template
• Sensitive detection of up to 5 targets in 1 tube

QuantiNova RT-PCR Kits (real-time RT-PCR kits) enable sensitive quantification of RNA targets by real-time one-step PCR using SYBR Green I or sequence-specific probes. A combination of various integrated safety features removes variables and prevents artifacts, ensuring reliable gene expression profiling. The combination of a unique two-phase hot-start and PCR buffer system in the ready-to-use master mix allows room-temperature setup and ensures highly sensitive qRT-PCR on any real-time cycler. The optional QuantiNova Internal Control RNA can be used to test successful reverse transcription and amplification. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors. Furthermore, the visual pipetting control can be used to monitor correct pipetting. The integrated gDNA reduction step in the QuantiNova Probe RT-PCR procedure prevents overquantification of transcripts caused by genomic DNA carryover.

The QuantiNova Multiplex RT-PCR Kit enables fast and reliable quantification of up to 5 RNA targets in a single tube by multiplex real-time RT-PCR. Our Q-Bond technology and optimized master mix promote ultrafast multiplex real-time RT-PCR within 1 hour. The combination of a unique two-phase hot-start procedure with our multiplex PCR buffer system ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization, and enables automated reaction setup at room temperature. The kit also provides protocols for extracted RNA and direct amplification from cultured cells, even down to a single cell. The optional QuantiNova Internal Control RNA can be used to monitor successful reverse transcription and amplification, and the visual pipetting control can be used to monitor correct pipetting.

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.

QuantiNova RT-PCR Kits contain optimized, ready-to-use master mixes for highly specific and sensitive real-time quantification of RNA targets using the fluorescent dye SYBR Green I or sequence-specific probes. The QuantiNova Probe RT-PCR Kit is designed for use with different types of sequence specific probes, including hydrolysis probes (e.g., TaqMan and other dual-labeled probes) and Scorpions primers. The kits come with a unique PCR buffer that contains a balanced combination of K⁺ and NH₄⁺ ions, which promotes specific primer annealing, enabling high specificity and sensitivity. In addition, the HotStaRT-Script enables cDNA synthesis from a wide range of RNA template amounts, while the QuantiNova DNA Polymerase provides a stringent hot-start procedure, preventing the formation of nonspecific products. The unique two-phase hot-start procedure allows reaction setup to remain stable for up to two hours at room temperature supporting automated reaction setup.

The built-in visual control allows you to check whether template has been added to the reaction and therefore prevents pipetting errors during reaction setup.

Detecting variations in cDNA synthesis allows you to check the reproducibility of your results. The newly developed internal control is a defined transcript (RNA molecule) that can be optionally added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors.

To obtain accurate results in real-time RT-PCR gene expression assays, it is important that only cDNA is amplified and detected. Interference by genomic DNA is prevented using the gDNA reduction step in QuantiNova Probe RT-PCR procedures (see figure “Increased reliability of gene expression results using gDNA reduction”). Time is saved and costs are reduced, since a separate DNase digestion is not required during or after purification of RNA samples. Also, designing RNA-specific primers or probes is not necessary.
The QuantiNova Multiplex RT-PCR Kit delivers highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization. The specially developed PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times. Amplifying reference and target genes in the same reaction increases reliability by minimizing well-to-well variations and handling errors. Additionally, an Internal Control RNA is provided and can be simultaneously detected to monitor successful reverse transcription and qPCR.

The QuantiNova SYBR Green and Probe RT-PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time consuming. Simply add primers, RNA template and RT mix to the ready-to-use RT-PCR or master mix and start the reaction. Follow the protocol in the QuantiNova SYBR Green RT-PCR Kit Handbook to get fast and reliable results on any real-time cycler.

The QuantiNova Multiplex RT-PCR Kit contains a ready-to-use 4x master mix, eliminating the need to optimize reaction and cycling conditions. Simply add the RT mix, up to 800 ng template RNA 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. ROX reference dye is also provided in a separate tube, so the concentration can be adjusted for your particular instrument. The kit also offers a simple and fast protocol for direct amplification from cultured cells. The recommended input amount ranges from 2000 cells down to a single cell, e.g., separated by serial dilution or using the QIAscout instrument. The QuantiNova Internal Control RNA (QN IC RNA) is an internal amplification control that can be optionally used to test for successful reverse transcription and amplification. It is detected as a 200 bp internal control in the yellow channel on the Rotor-Gene Q or in the VIC/HEX dye channel on other real-time PCR instruments when using the QuantiNova IC Probe Assay. Alternatively, it can be detected in the red channel on the Rotor-Gene Q or in the Cy5 dye channel on other real-time PCR instruments when using the QuantiNova IC Probe Assay Red 650.

The QuantiNova SYBR Green and Probe RT-PCR Kits can be used for gene expression analysis of RNA targets on any real-time cycler. This includes the Rotor-Gene Q and instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche and Agilent.

The QuantiNova Multiplex RT-PCR Kit can be used for multiplex gene expression analysis of RNA targets on any real-time cycler. To fully benefit from multiplexing, we recommend using an instrument that provides up to 5-plex capacity, such as the Rotor-Gene Q.