Rotor-Gene Multiplex RT-PCR Kit

Rotor-Gene Q での1ステップの超高速マルチプレックスリアルタイム RT-PCR

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
同一チューブ内で複数のRNAターゲットを高感度で検出
至適化実験なしに良好なマルチプレックスPCRを実現
発現量に差異がある複数のターゲットでも効率的に同時増幅

Product Details

Rotor-Gene Multiplex RT-PCR KitはRotor-Gene Qでの使用を目的としてデザインされ、配列特異的プローブを用いた1ステップのマルチプレックスリアルタイムRT-PCRで特異性の高い定量を高速に実現します。サイクラーの搭載波長によっては、同一チューブ中で最高4種類までのRNAターゲット（例；コントロール遺伝子1つと3つの標的遺伝子）を同時に定量できます。特別に至適化されたマスターミックスとRotor-Gene Q 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 Multiplex RT-PCR Kitは、発現量に大きな差異のあるduplex、triplex、4-plex アッセイにおいても、わずか10 pg のテンプレートから信頼できる定量を実現し、10 コピーのターゲットも検出できます（図 ""および""）。マルチプレックスアッセイにおいて全てのターゲットが同等の高いPCR 効率で増幅されます（図""）。そのため、目的遺伝子の発現量をコントロール遺伝子の発現量で補正する相対的定量において高い信頼性が得られます。

See figures

Reliable duplex analysis.

Efficient, sensitive duplex analysis.

Highly efficient 4-plex analysis.

Principle

別々の反応ではなく同一反応内でコントロール遺伝子と標的遺伝子を増幅することで、マニュアルでの作業によるエラーが最小限に抑えられ、遺伝子定量の信頼性が高くなります。Rotor-Gene Multiplex 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（フローチャート " QIAGEN multiplex kits"）。逆転写とPCRを同じ反応容器内で順次行なうことができ、1ステップのリアルタイムRT-PCRが可能です。逆転写反応の完了した反応液を他の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。

K⁺ イオンとNH₄⁺ イオンのバランスの取れた配合により、プライマーの特異的なアニーリングを促進するため、高感度で特異性の高い増幅が確実にできます。また、画期的なPCR添加剤である合成Factor MPは、難しいマルチプレックスPCRアプリケーション用に特別に開発されたもので、様々なアンプリコンを同一の反応で同様に高い効率で増幅できるようにします（図 " ユニークなPCRバッファー"）。

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"）。さらに最適化された逆転写酵素ミックスによりわずか15分で効率的なcDNAを行なう一方、厳密性の高いホットスタート酵素HotStarTaq Plus DNA PolymeraseはPCR 開始時の95℃、5分の短いインキュベーションで迅速に活性化されます。

2x Rotor-Gene Multiplex RT-PCR Kitの構成*
構成	特徴	利点
HotStarTaq Plus DNA Polymerase	95ºC、5分で活性化	室温で定量PCR反応のセットアップ
Rotor-Gene Multiplex RT-PCR Buffer	バランスの取れたNH₄⁺ イオンおよび K⁺ イオンの配合	プライマーの特異的アニーリングにより信頼できるPCR結果を実現
	合成Factor MP	4遺伝子まで同一チューブで信頼できるマルチプレックス解析が可能
	ユニークな Q-Bond 添加剤	PCR時間が短縮し、迅速に結果を得られ、1日あたりの反応数を増やせる
Rotor-Gene RT Mix	RNAに対して高い親和性を有する逆転写酵素を特別に配合	複雑な二次構造を取る場合でも、わずか15分でRNAを逆転写可能

See figures

QIAGEN multiplex kits.

Unique PCR buffer.

Fast primer annealing.

Procedure

即使用可能なマスターミックスにより、プライマーやプローブ濃度などの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、プライマープローブセット、添付の逆転写酵素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。製品のハンドブックには、推奨の色素リストやマルチプレックスRT-PCR 用の1種類のプロトコールなど、必要な情報が記載されています。

Applications

Rotor-Gene Multiplex RT-PCR Kitは、Rotor-Gene Q上で配列特異的プローブを用いて迅速な1ステップリアルタイムRT-PCR解析を行なうのに最適化されています。本キットは、Rotor-Gene 3000 およびRotor-Gene 6000にも適合します。最高4種類までのRNAターゲットを同一チューブ内で迅速かつ同時に定量できるため、処理数を増大し貴重なサンプルも節約できます。

Supporting data and figures

Reliable duplex analysis.

Duplex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for 28S rRNA (Cyanine 670 dye; data in inset) and PPIA (cyclophilin A, FAM dye). Triplicate reactions were run on the Rotor-Gene 3000 using leukocyte RNA as template (10-fold dilutions from 100 ng to 10 pg). The amplification plots for the duplex reactions (colored) overlapped with those for control singleplex reactions (gray), demonstrating the reliability of the duplex analysis.

Specifications

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

Resources

FAQ

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

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

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

The Rotor-Gene and QuantiFast Multiplex RT-PCR Kits allow reliable detection down to 10 target copies. Detection of lower copy numbers down to single copy level may also be possible; however, this depends on the stochastics when working with highly diluted samples. Additional optimization of primer/probe design is usually required.

FAQ ID -2144

The specific features of Rotor-Gene Kits and Rotor-Gene cyclers work synergetically to enable an ultrafast-cycling protocol. We do not guarantee that the performance of the Rotor-Gene Multiplex RT-PCR Kit with the same cycling protocol will be the same on other cyclers.

FAQ ID -2142

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line. Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

FAQ ID -9093

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

FAQ ID -9094

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098

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 -90990

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.

FAQ ID -9099