For ultrafast, one-step qRT-PCR using sequence-specific probes on Rotor-Gene cyclers

  • Optimized for ultrafast, reliable results on Rotor-Gene cyclers
  • Sensitive detection of even low copy numbers
  • Accurate detection of a wide range of template amounts
  • Specially formulated, ready-to-use master mix for fast cycling

The Rotor-Gene Probe RT-PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly sensitive quantification of RNA targets with real-time one-step RT-PCR using sequence-specific probes. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

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Product Cat. no. List price:
Rotor-Gene Probe RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene Probe RT-PCR Master Mix, 100 µl Rotor-Gene RT Mix, 2 x 2 ml RNase-Free Water
204574
$686.00
The Rotor-Gene Probe RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

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Sensitive detection using a sequence-specific probe.|Specific primer annealing.|Fast primer annealing.|
Tenfold dilutions of human leukocyte RNA (100 ng to 1 ng) were used as template in real-time one-step RT-PCR. Triplicate reactions were run using a TaqMan Gene Expression Assay for IL12RB1 (interleukin 12 receptor, beta 1). Greater sensitivity (i.e., lower CT values) was achieved with [A] the Rotor-Gene Q and Rotor-Gene Probe RT-PCR Kit than with [B] an instrument and kit from Supplier AII.|Cations in the Rotor-Gene Q PCR buffer increase specific primer annealing. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in Rotor-Gene Probe RT-PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|
Performance
With the Rotor-Gene Probe RT-PCR Kit, low-abundance transcripts are reliably quantified (see figure "Sensitive detection using a sequence-specific probe").
Principle

The Rotor-Gene Probe RT-PCR Kit enables reliable quantification of RNA templates on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. The kit is designed for use with hydrolysis probes (e.g., TaqMan® probes). RNA is used as the template in a reaction where reverse transcription and PCR take place sequentially in the same reaction vessel. Since it is not necessary to transfer the finished RT reaction to another tube for PCR, the real-time RT-PCR procedure is streamlined, making high-throughput analysis possible.

Highly specific amplification is assured through a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that considerably shortens cycler run times (see figure "Fast primer annealing").

Components of 2x Rotor-Gene Probe RT-PCR Kit*
Component Features Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
Rotor-Gene Probe RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable qPCR results
Unique Q-Bond additive Faster PCR run times enable faster results and more reactions per day
Rotor-Gene RT Mix   Special blend of reverse transcriptases with a high affinity for RNA     RNA can be transcribed in just 10 minutes, even through complex secondary structures
* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).
Procedure

The ready-to-use Rotor-Gene Probe RT-PCR Master Mix eliminates the need for optimization of reaction and cycling conditions. Just add template RNA, primers, probe, and the supplied reverse transcriptase mix to the master mix and program the cycler. Instructions are provided in the detailed handbook supplied with the kit.

Hydrolysis probes (e.g., TaqMan® probes) can be used in combination with the Rotor-Gene Probe RT-PCR Kit on the Rotor-Gene Q for fast and sensitive quantification — simply add the primer-probe mix and template to the master mix.

Applications

The Rotor-Gene Probe RT-PCR Kit is well suited for use in fast, real-time one-step RT-PCR of RNA targets using sequence specific probes on the Rotor-Gene Q. It is also compatible with Rotor-Gene 3000 and Rotor-Gene 6000 cyclers.

Feature
Specifications
Applications Real-time quantification of RNA targets
Description For ultrafast quantitative real-time one-step RT-PCR using sequence-specific probes
Reaction type Real-time one-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA
Single or multiplex Single
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
With or without ROX Without ROX dye

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Brochures & Guides
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Kit Handbooks
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For fast real-time PCR, two-step RT-PCR, and one-step RT-PCR using sequence-specific probes on Rotor-gene cyclers.
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Rotor-Gene Probe PCR Kit
Rotor-Gene Probe RT-PCR Kit

配列特異的なプローブとRotor-Gene サイクラーを用いた迅速なリアルタイムPCR、2ステップRT-PCR、1ステップRT-PCR
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