Rotor-Gene SYBR® Green RT-PCR Kit

For ultrafast, one-step qRT-PCR gene expression analysis using SYBR Green I 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
  • Guaranteed performance combined with QuantiTect Primer Assays

The Rotor-Gene SYBR Green RT-PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly specific quantification of RNA targets with real-time one-step RT-PCR using SYBR Green I detection. 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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Rotor-Gene SYBR Green RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene SYBR Green RT-PCR Master Mix, 100 µl Rotor-Gene RT Mix, 2 x 2 ml RNase-Free Water
 204174
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The Rotor-Gene SYBR® Green 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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Specific detection without the need for optimization.|Specific primer annealing.|Fast primer annealing.|
Tenfold dilutions of human leukocyte RNA (100 ng to 10 pg) were used as template in SYBR® Green-based real-time one-step RT-PCR. Duplicate reactions were run using the QuantiTect Primer Assay for BCL2 (B-cell CLL/lymphoma 2). [A] The Rotor-Gene Q and Rotor-Gene SYBR® Green RT-PCR Kit provided sensitive detection from 10 pg RNA and amplification of specific PCR product (melting curve shown in inset). [B] In contrast, an instrument and kit from Supplier R provided detection only after optimization of Mg2+ concentration. However, the limit of detection was 100 pg RNA and coamplification of nonspecific products was observed (melting curve shown in inset).|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 SYBR® Green 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
When the Rotor-Gene SYBR Green RT-PCR Kit is used together with QuantiTect Primer Assays, highly sensitive quantification of specific PCR products is achieved without the need for optimization (see figures "Specific detection without the need for optimization").
Principle

The Rotor-Gene SYBR Green RT-PCR Kit enables reliable real-time RT-PCR quantification on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. Real-time one-step RT-PCR is carried out, which means that RNA is used as 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.

The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. 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 SYBR Green RT-PCR Kit*
ComponentFeatures Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
Rotor-Gene SYBR Green 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
SYBR Green I dye Yields a strong fluorescent signal upon binding double-stranded DNA Highly sensitive quantification
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

A ready-to-use master mix eliminates the need for optimization of reaction and cycling conditions. Simply add template RNA, primers, 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.

For gene expression analysis using real-time one-step RT-PCR, the combination of the Rotor-Gene SYBR Green RT-PCR Kit with QuantiTect Primer Assays and the Rotor-Gene Q provides a complete, ready-to-run solution. QuantiTect Primer Assays are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.

Applications

The Rotor-Gene SYBR Green RT-PCR Kit provides rapid real-time quantification of RNA targets on the Rotor-Gene Q. The kits are also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000.

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Feature
Specifications
Applications Real-time quantification of RNA targets
Description For ultrafast quantitative real-time one-step RT-PCR using SYBR Green I
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 SYBR Green I
Thermal cycler Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
With or without ROX Without ROX dye

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Brochures & Guides
1
(EN) - Rotor-Gene Q - Pure Detection
Now with even more applications!
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Kit Handbooks
2
(EN) - Rotor-Gene SYBR Green Handbook
For fast real-time PCR, two-step RT-PCR, and one-step RT-PCR using SYBR Green I on Rotor-Gene cyclers
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(EN) - QuantiTect Primer Assay Handbook
For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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