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QuantiNova Probe RT-PCR Kit

For one-step qRT-PCR using sequence-specific probes for gene expression analysis

 

  • Unique two-phase hot-start procedure for room-temperature setup
  • Visual pipetting control, resulting in fewer pipetting errors
  • gDNA reduction for improved quantification accuracy
  • Internal control for positive in-process verification of successful RT-PCR
  • Duplex capability, enabling inclusion of internal control or reference gene
  • Accurate quantification over several logs of template
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.
Cat No./ID: 205813
QuantiNova IC Probe Assay (200)
$173.00
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400 µl primer / probe mix (10x) for 200 reactions, detects Internal Control RNA; use with QuantiNova Probe PCR Kit or QuantiNova Probe RT-PCR Kit
Cat No./ID: 208352
QuantiNova Probe RT-PCR Kit (100)
$185.00
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For 100 x 20 µl reactions: 1 ml QuantiNova Probe RT-PCR Master Mix, 20 µl QuantiNova Probe RT Mix, 20 µl Internal Control RNA, 500 µl Yellow Template Dilution Buffer, 250 µl ROX Reference Dye, 1.9 µl RNase-Free Water
Cat No./ID: 208354
QuantiNova Probe RT-PCR Kit (500)
$743.00
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For 500 x 20 µl reactions: 3 x 1.7 ml QuantiNova Probe RT-PCR Master Mix, 100 µl QuantiNova Probe RT Mix, 100 µl Internal Control RNA, 500 µl Yellow Template Dilution Buffer, 1 ml ROX Reference Dye, 2 x 1.9 µl RNase-Free Water
Cat No./ID: 208356
QuantiNova Probe RT-PCR Kit (2500)
$2,969.00
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For 2500 x 20 µl reactions: 15 x 1.7 ml QuantiNova Probe RT-PCR Master Mix, 5 x 100 µl QuantiNova Probe RT Mix, 5 x 100 µl Internal Control RNA , 5 x 500 µl Yellow Template Dilution buffer, 5 x 1 ml ROX Reference Dye, 10 x 1.9 µl RNase-Free Water
Cat No./ID: QT02589307
QuantiNova IC SYBR Green Assay
QuantiTect Primer Assay for SYBR-based detection of QuantiNova Internal Control RNA; available via GeneGlobe (sufficient for approx. 500 x 20 µl rxn). For use with QuantiNova SYBR Green PCR Kit or QuantiNova SYBR Green RT-PCR Kit
The QuantiNova Probe RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
0
One-step RT-PCR with comparable performance to two-step RT-PCR - B.

Tenfold serial dilutions of RNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for ILR2 (interleukin 1 receptor, type II). One-step RT-PCR was performed using the QuantiTect Probe RT-PCR Kit (PCR efficiency: 104%).

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Increased reliability of gene expression results using gDNA reduction.
Total HeLa RNA (10 ng/rnx) samples were spiked with increasing amounts of human genomic DNA. Expression of MGAT1 was analyzed by qRT-PCR. MGAT1 is a single-exon gene that does not allow the discrimination of RNA and gDNA by assay design. Without the gDNA reduction step, Cq values decreased linearly, falsely indicating an increase in expression rate. In contrast, the expression levels remained similar when using the gDNA reduction step.
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Principle of the novel QuantiNova two-phase hot-start mechanism.
At ambient temperature the HotStaRTScript is inhibited by the RT-Blocker and the QuantiNova DNA Polymerase is kept inactive by QuantiNova Antibody and QuantiNova Guard. At 45°C the RT is activated while the QuantiNova DNA polymerase remains inactive. At 95°C the RT enzyme is denatured and the DNA polymerase is activated.
3
Convenient room-temperature reaction setup without compromising performance.
Real-time RT-PCRs including master mix, template RNA, primers and probes for [A] GAPDH, [B] MYC and [C] HSP90AA1 were left at room temperature (22°C) for 1 h and 2 h. These samples were run in parallel to freshly prepared reactions (set up on ice) on the Bio-Rad CFX 384 instrument. Duplicate reactions of 3 different template amounts were tested. The Cq values provided by the QuantiNova Probe RT-PCR Kit did not vary after extended storage of the samples at room temperature.
4
Superior sensitivity of the QuantiNova Probe RT-PCR Kit.
The performance of the QuantiNova Probe RT-PCR Kit was compared to a probe RT-PCR kit from supplier B on [A] a Bio-Rad CFX Connect cycler and [B] a probe RT-PCR kit from supplier L on a ViiA7 cycler. Reactions were run in duplicate using 10-fold dilutions of HeLa total RNA (100 ng – 10 pg) using TaqMan® assays for [A] MYC or [B] CDKNB1. Results show a high correlation between kits for higher template amounts; however, sensitivity was higher with the QuantiNova Probe RT-PCR Kit at 100 pg and 10 pg of total RNA (red arrows) compared to kits from supplier B and L.
5
Accurate reaction setup indicated by the built-in pipetting control.
The master mix contains an inert blue dye. Combined with QuantiNova Yellow Template Dilution Buffer, the resulting solution turns green, indicating that the reaction was set up correctly.
Performance
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”).
Principle
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.
Procedure
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.
Applications
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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