QuantiNova Probe RT-PCR Kit
For one-step qRT-PCR using sequence-specific probes for gene expression analysis
The QuantiNova Probe RT-PCR Kit enables sensitive quantification of RNA targets by real-time one-step PCR using 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 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. What's more, the visual indicator reduces pipetting error while the integrated gDNA reduction step prevents overquantification of transcripts caused by genomic DNA carryover.
The QuantiNova Probe 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 45°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 45°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, that stabilizes the complex. This improves the stringency of the PCR hot-start procedure and prevents extension of non-specifically annealed primers and primer–dimers. Within 5 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 (see figure “Convenient room-temperature reaction setup without compromising performance”).
The gDNA reduction step 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 master mix supplied with the QuantiNova Probe 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 (see figure “Superior sensitivity of the QuantiNova Probe RT-PCR Kit”).
The QuantiNova Probe RT-PCR Kit contains an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using sequence-specific probes. The 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 kit comes with a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote 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.
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. 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 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.
The QuantiNova Probe RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time consuming. Simply add primers, probe and RNA template to the ready-to-use RT-PCR master mix and start the reaction. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler.
The QuantiNova Probe RT-PCR Kit 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.
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