Rotor-Gene SYBR^® Green PCR Kit

For ultrafast, real-time PCR and two step RT-PCR using SYBR Green on Rotor-Gene cyclers

Products

The Rotor-Gene SYBR^® Green PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Features

Specific and sensitive detection of even low-copy targets
Accurate detection of a wide range of template amounts
Specially formulated master mix for reliable results without optimization
Guaranteed performance combined with QuantiTect Primer Assays

Product Details

The Rotor-Gene SYBR Green PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly specific quantification of gDNA and cDNA targets with quantitative real-time PCR or two-step RT-PCR using SYBR Green I detection. Outstanding qPCR 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

The Rotor-Gene SYBR Green PCR Kit is well suited for use in gene expression analysis of cDNA targets (see figures " Highly specific and sensitive detection" and " Reliable and specific detection down to 10 copies").

When QuantiTect Primer Assays are used together with the Rotor-Gene SYBR Green PCR Kit, highly sensitive quantification of specific PCR products is achieved without the need for optimization (see table).

Superior performance in RT-PCR with SYBR Green
	QIAGEN		Supplier A_II
	C_T	Mean deviation	C_T	Mean deviation
BAX (BCL2-associated X protein)	24.84	0.05	29.57	0.46
BCL2 (apoptosis gene)	26.96	0.05	32.83	0.29
MYC (proto-oncogene)	28.42	0.21	35.26	0.72
b-Actin (housekeeping gene)	20.24	0.03	24.39	0.12

See figures

Highly specific and sensitive detection.

Reliable and specific detection down to 10 copies.

Principle

The Rotor-Gene SYBR Green PCR Kit enables rapid and reliable real-time PCR quantification on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. 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 enables cycler run times of as low as 45 minutes (see figure " Fast primer annealing").

Components of 2x Rotor-Gene SYBR Green 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 PCR Buffer	Balanced combination of NH₄⁺ and K⁺ ions	Specific primer annealing ensures reliable qPCR results
Rotor-Gene SYBR Green 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

See figures

Specific primer annealing.

Fast primer annealing.

Procedure

The Rotor-Gene SYBR Green PCR Master Mix eliminates the need for optimization of reaction and cycling conditions. Simply add DNA template DNA and primers 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 two-step RT-PCR, the combination of the Rotor-Gene SYBR Green PCR Kit, QuantiTect Reverse Transcription Kit, QuantiTect Primer Assays, and Rotor-Gene Q provides a complete, ready-to-run solution. The QuantiTect Reverse Transcription Kit delivers fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination. 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 PCR Kit provides rapid real-time quantification of cDNA and gDNA targets on the Rotor-Gene Q. The Kits are also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000.

For ultrafast, one-step qRT-PCR gene expression analysis of RNA targets using SYBR Green I on Rotor-Gene cyclers, use the Rotor-Gene SYBR Green RT-PCR Kit.

Supporting data and figures

Highly specific and sensitive detection.

Real-time two-step RT-PCR was carried out using tenfold dilutions of human leukocyte cDNA (10 ng to 0.1 ng) and a QuantiTect Primer Assay for BCL2 (B-cell CLL/lymphoma 2). Reactions were run in triplicate using either [A] the Rotor-Gene SYBR^® Green PCR Kit and Rotor-Gene Q or [B] a kit and cycler from Supplier A_II. The Rotor-Gene system provided lower C_T values and greater reproducibility within triplicates. Melting curve analysis (see insets) showed a single peak, demonstrating specific amplification of the target.

Specifications

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

Resources

FAQ

Based on our extensive and successful testing of many QuantiTect Primer Assays with Rotor-Gene SYBR Green PCR Kits, we guarantee this.

FAQ ID -2124

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 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

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

Assuming 100% amplification efficiency, each step increase in Ct value represents a doubling in the amount of qPCR template. Therefore, evaluating the difference in Ct values between the qPCR assay, and its matching NRT control, leads to the following predictions:

CtNRT - Ct+RT	Fraction of gene expression signal due to contaminating DNA	Percentage of gene expression signal due to contaminating DNA
1	(1/21) = 1/2	50%
2	(1/22) = 1/4	25%
3	(1/23) = 1/8	13%
4	(1/24) = 1/16	6%
5	(1/25) = 1/32	3%

FAQ ID -2688

There are several manufacturers of high-quality real-time cyclers. These include QIAGEN's Rotor-Gene Q, Applied Biosystems, BioRad, Stratagene, Eppendorf, Roche, TaKaRa, Fluidigm, and Cepheid. The important thing to keep in mind is that, once you select an instrument to use, you must use compatible Rotor-Gene Discs and tubes, 96 or 384 well plates, and qPCR master mixes that are optimized for use in that particular instrument. For example, QIAGEN's Rotor-Gene SYBR Green, Probe, and Multiplex real-time master mixes.

FAQ ID -2670

Dissociation curves are carried out at the end of a PCR experiment by following a 3-step procedure.

First, all the components are denatured at 95°C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95°C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve.

By analyzing the first derivative of such a curve, you can readily assess the homogeneity of the PCR products, including the presence of primer–dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry due to this reagent's lack of sequence specificity.

FAQ ID -2678

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

We have performed numerous tests comparing the performance of Rotor-Gene SYBR Green PCR Kits and QuantiTect Kits with QuantiTect Primer Assays. Due to the optimized ion concentrations in the PCR buffers, both perform equally well with QuantiTect Primer Assays and do not require any adjustment of primer concentration.

FAQ ID -2123

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

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

FAQ ID -2690