Rotor-Gene Probe RT-PCR Kit

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

Features

  • 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
Rotor-Gene Probe RT-PCR Kit (400)

Cat. No. / ID: 204574

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
$707.00
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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.

Product Details

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.

 

IMPORTANT NOTE: This product is phasing-out until June 30, 2021, and afterward, available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.

 

For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

Performance

With the Rotor-Gene Probe RT-PCR Kit, low-abundance transcripts are reliably quantified (see figure " Sensitive detection using a sequence-specific probe").
See figures

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).
See figures

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.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsReal-time quantification of RNA targets
Reaction typeReal-time one-step RT-PCR
Real-time or endpointReal-time
Thermal cyclerRotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
SYBR Green I or sequence-specific probesSequence-specific probes
Sample/target typeRNA
Single or multiplexSingle
DescriptionFor ultrafast quantitative real-time one-step RT-PCR using sequence-specific probes
With or without ROXWithout ROX dye

Resources

Kit Handbooks (1)
For fast real-time PCR, two-step RT-PCR, and one-step RT-PCR using sequence-specific probes on Rotor-gene cyclers.
Brochures & Guides (1)
Now with even more applications!

FAQ

Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

FAQ ID -2371
Do the master mixes in Rotor-Gene Kits contain dUTP to allow UNG pretreatment?

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

 

 

FAQ ID -2117
Why is the initial activation step different for Rotor-Gene Probe, SYBR Green and Multiplex Kits?

The buffer composition, which affects the initial reactivation of HotStarTaq Plus DNA Polymerase, has been optimized for each respective Rotor-Gene Kit.

 

FAQ ID -2118
How important is the RNA purification process, for obtaining reliable qRT-PCR results?

The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment.

Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It is also a source of false positive signals in RT-PCR experiments.

RNase contamination degrades RNA samples whichcauses low signal and false-negative results in PCR.

Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

For fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits like the RNeasy Mini Kit, the RNeasy Plus Universal Kit, or the RNeasy FFPE Kit.

FAQ ID -2655
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling?

Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.

FAQ ID -1430
Are Rotor-Gene Kits compatible with reaction setup using the QIAgility instrument?

The majority of the Rotor-Gene Kit data shown in our literature has been generated with the help of the QIAgility instrument. We did not observe any problems during the pipetting steps.

 

FAQ ID -2116
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

 

FAQ ID -2119
Will Rotor-Gene Kits also work on the Rotor-Gene 6000 and 3000 cyclers?

Yes. Rotor-Gene Kits will also work on the Rotor-Gene 6000 and Rotor-Gene 3000 PCR cyclers with the cycling conditions specified in the Rotor-Gene kit handbooks.

 

FAQ ID -2121