Rotor-Gene SYBR^® Green PCR Kit

SYBR Green を用いたRotor-Geneサイクラーでの超高速リアルタイムPCRおよび2ステップRT-PCR

Products

Rotor-Gene SYBR^® Green PCR Kit は分子生物学実験用です。疾病の診断、治療または予防の目的に使用することはできません。

Features

低コピーターゲットでも特異的で高感度な検出
幅広いテンプレート量で正確に検出
至適化なしに信頼できる結果を実現する特別調製のマスターミックス
QuantiTect Primer Assayと組み合わせて最適な結果

Product Details

Rotor-Gene SYBR Green PCR KitはRotor-Gene QおよびRotor-Geneサイクラーでの使用を目的としてデザインされ、SYBR Green I検出を用いたリアルタイム定量PCRあるいは2ステップリアルタイムRT-PCR で特異性の高いｇDNAやcDNAターゲットの高速で高感度な定量を実現します。特別に至適化されたマスターミックスとRotor-Geneサイクラーの遠心エアコントロール方式の組み合わせにより、最高の性能を実現します。マスターミックスは2～8 ℃で保存でき、簡便に取り扱えます。

Performance

Rotor-Gene SYBR Green PCR KitはcDNAターゲットの遺伝子解析で使用するのに最適です（図“ 特異性と感度の高い検出” および“ 10コピーのターゲットを確実かつ高い特異性で検出”)。

QuantiTect Primer AssaysはRotor-Gene SYBR Green PCR Kit と組み合わせると、至適化なしに特異的なPCR 産物の高感度な定量が行なえます（表参照）。

SYBR Green を用いたRT-PCR で卓越した性能
	QIAGEN		A_II社
	C_T	平均偏差	C_T	平均偏差
BAX (BCL2結合Xタンパク質)	24.84	0.05	29.57	0.46
BCL2 (アポトーシス遺伝子)	26.96	0.05	32.83	0.29
MYC (がん原遺伝子)	28.42	0.21	35.26	0.72
β-アクチン（ハウスキーピング遺伝子）	20.24	0.03	24.39	0.12

See figures

Highly specific and sensitive detection.

Reliable and specific detection down to 10 copies.

Principle

Rotor-Gene SYBR Green PCR Kit は Rotor-Gene Q において反応条件やサイクリング条件の至適化なしに信頼できる迅速なリアルタイムPCR定量を実現します。マスターミックス中の蛍光色素SYBR Green Iは、ターゲットに特異的な標識プローブを合成しなくても多数の異なるターゲットを解析できます。特異的なプライマーアニーリングを促進するK⁺とNH₄⁺イオンのバランスの取れた配合により、特異性の高い増幅が確保され、PCRの高い特異性と感度を実現します (図 “ プライマーの特異的なアニーリング”)。画期的なPCR 添加物であるQ-Bond により性能を損なうことなく高速サイクリングが行なえ、ラン時間はわずか45 分です（図“ プライマーの高速なアニーリング”）。

2x Rotor-Gene SYBR Green PCR Kitの成分*
成分	特長	利点
HotStarTaq Plus DNA Polymerase	95℃、5分の活性化	室温でのｑPCRのセットアップ
Rotor-Gene SYBR Green PCR Buffer	NH₄⁺とK⁺イオンの配合バランス	特異性の高いプライマーのアニーリングで信頼性の高いｑPCR結果
Rotor-Gene SYBR Green PCR Buffer	ユニークな添加物Q-Bond	より迅速なPCRランタイムでより早く結果が得られ、一日あたりの反応数も増大
SYBR Green I 色素	ニ本鎖DNAに結合して強い蛍光シグナルを発生	感度の高い定量

See figures

Specific primer annealing.

Fast primer annealing.

Procedure

Rotor-Gene SYBR Green PCR Master Mixにより、反応条件やサイクリング条件の至適化が不要です。DNAテンプレートとプライマーをマスターミックスに添加し、サイクラーをプログラミングします。キットに添付のハンドブックに詳細な説明が記載されています。

２ステップリアルタイムRT-PCRを用いた遺伝子発現解析において、 Rotor-Gene SYBR Green PCR Kit、 QuantiTect Reverse Transcription Kit、QuantiTect Primer Assays、Rotor-Gene Q の組み合わせは、最適化なしに開始できるトータルソリューションです。QuantiTect Reverse Transcription Kitは、ゲノムDNAコンタミの除去を組み合わせたcDNA合成をわずか20分で行ないます。QuantiTect Primer Assay は、ヒト、マウス、ラット、その他の生物種のほとんどの遺伝子に対応するバイオインフォマティックスで検証済みのプライマーセットです。アッセイはGeneGlobeウェブポータルサイトで容易に注文できます。

Applications

Rotor-Gene SYBR Green PCR KitはRotor-Gene Q上でcDNAやgDNAターゲットの迅速なリアルタイム定量に最適です。本キットはまたRotor-Gene 3000およびRotor-Gene 6000でも使用可能です。

Rotor-Geneサイクラ―上でSYBR Green Iを用いてRNAターゲットを超迅速な1ステップでqRT-PCRで遺伝子解析する場合には、Rotor-Gene SYBR Green RT-PCR Kitをご使用ください。

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
Thermal cycler
Reaction type
SYBR Green I or sequence-specific probes
Description
Single or multiplex
Real-time or endpoint
With or without ROX
Sample/target type

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