Rotor-Gene SYBR^® Green RT-PCR Kit

SYBR Green Iを用いたRotor-Geneサイクラーでの超高速1ステップリアルタイムRT-PCR遺伝子発現解析

Features

Rotor-Gene サイクラーでの信頼できる結果を超高速で実現するために最適化済み
低コピー数の標的遺伝子でも高感度な検出
幅広いテンプレート量で正確に検出
高速サイクリング用に特別に調製された即使用可能なマスターミックス
QuantiTect Primer Assayと組み合わせて最適な結果

Product Details

Rotor-Gene SYBR Green RT-PCR Kit は Rotor-Gene Qおよび他の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を用いた1ステップリアルタイムRT-PCR で特異性の高いRNAターゲットの高速定量を実現します。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 RT-PCR KitをQuantiTect Primer Assaysと組み合わせて使用すると、至適化なしに特異的なPCR産物の高感度な定量を実現します（図 “”)。

See figures

Specific detection without the need for optimization.

Principle

Rotor-Gene SYBR Green RT-PCR Kit は Rotor-Gene Q において信頼できるリアルタイムRT-PCR定量を実現します。反応条件やサイクリング条件の至適化は不要です。逆転写反応と増幅を同一チューブ中で行なう1ステップリアルタイムRT-PCRのテンプレートとしてRNAを使用します。逆転写反応の完了したRT反応液を他のPCR用チューブに移し替える必要がないのでリアルタイムRT-PCR操作を効率的に行なえ、ハイスループット解析が可能です。

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、特異性の高い増幅か確保され、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、サイクラーのランニング時間は大幅に短縮されます（図“ プライマーの高速なアニーリング”)。

2x Rotor-Gene SYBR Green RT-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 RT-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 RT-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
Rotor-Gene RT Mix	RNA親和性の高い逆転写酵素を特別に配合	複雑な二次構造を持つRNAでもわずか10分で逆転写可能

See figures

Specific primer annealing.

Fast primer annealing.

Procedure

即使用可能なマスターミックスにより、反応条件やサイクリング条件の至適化が不要です。RNAテンプレート、プライマー、添付の逆転写反応ミックス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。

1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 RT-PCR 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 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 RT-PCR KitはRotor-Gene Q上でRNA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。

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

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