Rotor-Gene SYBR^® Green PCR Kit

在Rotor-Gene PCR仪上使用SYBR Green I通过两步法qRT-PCR超快速进行基因表达分析

Products

Rotor-Gene SYBR^® Green PCR Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。

Features

特异性检测低拷贝靶基因
精确检测广泛模板量范围内的基因
特制的即用型预混液，用于快速循环
配合QuantiTect Primer Assays使用，可保证性能

Product Details

Rotor-Gene SYBR Green PCR Kit专用于Rotor-Gene Q实时荧光定量PCR分析仪和其他的Rotor-Gene PCR仪，使用SYBR Green I对cDNA靶基因进行超快速、高特异性的real-time PCR和两步法RT-PCR分析。经优化的预混液与独特的Rotor-Gene PCR仪配合使用保证了qPCR良好的性能。预混液可贮存在2–8°C，方便使用。

Performance

Rotor-Gene SYBR Green PCR Kit特别适用于对cDNA靶基因进行基因表达分析（参见"Highly specific and sensitive detection"和"Reliable and specific detection down to 10 copies"）。

QuantiTect Primer Assays与Rotor-Gene SYBR Green PCR Kit配合使用，可高灵敏度定量检测特异性PCR产物，无需经过优化（参见下表）。

使用SYBR Green在RT-PCR中获得优越性能
	QIAGEN		Supplier A_II
	C_T	平均偏差	C_T	平均偏差
BAX (BCL2-associated X protein)	24.84	0.05	29.57	0.46
BCL2 (凋亡基因)	26.96	0.05	32.83	0.29
MYC (原癌细胞)	28.42	0.21	35.26	0.72
b-Actin (管家基因)	20.24	0.03	24.39	0.12

Principle

Rotor-Gene SYBR Green PCR Kit可在Rotor-Gene Q实时荧光定量PCR分析仪上进行快速、可靠的real-time PCR定量分析，无需优化反应及循环条件。混合液中的荧光染料SYBR Green I能用于分析多种不同的靶基因而无需合成靶标特异性标签探针。预混液中浓度平衡的K⁺和NH₄⁺离子组合保证了高特异性扩增，使引物退火高度特异性，获得高PCR特异性和灵敏性（参见"Specific primer annealing"）。使用创新的PCR辅助剂Q-Bond使循环时间降低至45分钟，达到快速分析且不降低性能（参见"Fast primer annealing"）。

2x Rotor-Gene SYBR Green PCR Kit的组分*
组分	特点	优势
HotStarTaq Plus DNA Polymerase	在95ºC下5分钟活化	室温下进行qPCR反应体系构建
Rotor-Gene SYBR Green PCR Buffer	浓度平衡的K⁺和NH₄⁺离子组合	引物特异性结合保证可靠的qPCR结果
Rotor-Gene SYBR Green PCR Buffer	独特的Q-Bond添加剂	快速的PCR运行时间可获得较快的结果和每天更多的反应次数
SYBR Green I染料	与双链DNA结合产生较强的荧光信号	高度灵敏的定量检测

Procedure

Rotor-Gene SYBR Green PCR Master Mix无需优化反应及循环条件。只需将cDNA模板DNA与引物加入到预混液中并设置循环程序。在试剂盒使用手册中有详细说明。

对于基于real-time两步法RT-PCR的基因表达分析，Rotor-Gene SYBR Green PCR Kit、QuantiTect Reverse Transcription Kit、QuantiTect Primer Assays及Rotor-Gene Q实时荧光定量PCR分析仪提供了完成的即用型解决方案。QuantiTect Reverse Transcription Kit可以在20分钟内快速合成cDNA并完全去除基因组DNA污染。QuantiTect Primer Assays为覆盖全基因组，经功效验证的预制引物对，用于检测来自人、小鼠、大鼠和其他物种的转录本。QuantiTect Primer Assays可以便利的在GeneGlobe网上订购。

Applications

Rotor-Gene SYBR Green PCR Kit适用于在Rotor-Gene Q实时荧光定量PCR分析仪上对cDNA靶基因进行快速real-time定量分析。该试剂盒与Rotor-Gene 3000和Rotor-Gene 6000兼容。

可使用Rotor-Gene SYBR Green RT-PCR Kit在Rotor-Gene PCR仪上通过SYBR Green I对目标RNA进行超快速、一步法qRT-PCR基因表达分析。

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.

Reliable and specific detection down to 10 copies.

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