Rotor-Gene SYBR^® Green RT-PCR Kit

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

Product Details

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.

IMPORTANT NOTE: As announced earlier, the production of the Rotor-Gene kits has been discontinued since mid-2021. Hence, these products will be 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

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 "").

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*
Component	Features	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
Rotor-Gene SYBR Green RT-PCR Buffer	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

See figures

Specific primer annealing.

Fast primer annealing.

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.

Supporting data and figures

Specific detection without the need for optimization.

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).

Specifications

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

Resources

FAQ

Yes, please follow the Supplementary Protocols at the links below:

Rotor-Gene SYBR Green PCR Kit:

'Rotor-Gene software setup for the Rotor-Gene SYBR Green PCR Kit' (PCR106)

Rotor-Gene SYBR Green RT-PCR Kit:

'Rotor-Gene software setup for the Rotor-Gene SYBR Green RT-PCR Kit' (PCR107)

FAQ ID -2104

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.

QuantiTect Primer Assays are primer sets for use in SYBR Green based real-time RT-PCR analysis.
QuantiFast Probe Assays are primer-probe sets for use in dual-labeled 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

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

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
Use only fresh PCR-grade reagents and disposable labware.
Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.

FAQ ID -2654

QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.

For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.

FAQ ID -1141

This type of cycling allows a significant reduction in cycling time for Rotor-Gene SYBR Green PCR Kits. It is more effective than reducing the individual times for annealing and extension.

FAQ ID -2122

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

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

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

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

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

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