Rotor-Gene SYBR^® Green RT-PCR Kit

在Rotor-Gene PCR仪上使用SYBR Green I通过一步法qRT-PCR超快速进行基因表达分析

Features

经优化，可在Rotor-Gene PCR仪上超快速获得可靠的结果
灵敏检测低拷贝靶基因
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 RT-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RNA靶基因进行超快速、高特异性的一步法real-time RT-PCR。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

QuantiTect Primer Assays与Rotor-Gene SYBR Green RT-PCR Kit联合使用，高灵敏度定量特异性PCR产物无需经过优化（参见""）。

See figures

Specific detection without the need for optimization.

Principle

Rotor-Gene SYBR Green RT-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 RT-PCR定量分析，无需优化反应及循环条件。该产品利用一步法real-time RT-PCR技术，RNA在反应中作为模板，在同一反应体系中实现逆转录和PCR反应。因为无需将逆转录反应后的反应体系转移到另外的管中进行PCR反应，整个real-time RT-PCR反应十分高效，适用于高通量分析。

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 RT-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 RT-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 RT-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 dye	与双链DNA结合产生较强的荧光信号	高度灵敏的定量检测
Rotor-Gene RT Mix	对RNA有高度亲和性的逆转录酶混合液	即便具有复杂的二级结构，RNA在10分钟内即可完成逆转录

See figures

Specific primer annealing.

Fast primer annealing.

Procedure

即用型预混液无需优化反应及循环条件。只需将RNA模板、引物和逆转录酶混合液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一步法RT-PCR的基因表达分析，Rotor-Gene SYBR Green RT-PCR 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 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 RT-PCR Kit适用于在Rotor-Gene Q实时荧光定量PCR分析仪上对RNA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。

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