QuantiFast Multiplex RT-PCR Kits

配列特異的なプローブを用いた遺伝子発現解析用高速1ステップマルチプレックスリアルタイム定量RT-PCR

Products

QuantiFast Multiplex RT-PCR Kitsは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

QuantiFast Multiplex RT-PCR +R Kit (2000)

Cat. No. / ID: 204956

For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 500 µl QuantiFast RT Mix, 1.05 ml ROX Dye Solution, 1 x 20 ml RNase-Free Water

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QuantiFast Multiplex RT-PCR Kit (400)

Cat. No. / ID: 204854

IMPORTANT NOTE: As announced earlier, the production of this kit has been discontinued since mid-2021. Hence, this product will be available only until stocks last. For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex RT-PCR Master Mix (with ROX dye), 100 µl QuantiFast RT Mix, 2 x 2 ml RNase-Free Water

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This kit is being phased out. We recommend switching to the QuantiNova successor product. For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

特徴

同一ウェル内で複数のターゲットRNAを高感度で検出
時間を最大50％まで短縮し迅速な結果
至適化実験なしに良好なマルチプレックスRT-PCRを実現
わずかなターゲット量の違いも正確に判別
リファレンス遺伝子と3種類までのターゲットを同一チューブ内で検出可能

製品詳細

QuantiFast Multiplex RT-PCR Kit は、1ステップのマルチプレックスリアルタイム定量RT-PCR により、同一チューブ中で最高4種類のRNAターゲットを高速かつ確実に定量します。特殊な割合で配合された逆転写酵素により迅速で効率的なcDNA合成を実現します。Q-bondテクノロジーと至適化済みのマスターミックスにより、短いRamping Time機能を持つ高速サイクラーだけではなくほとんどのサイクラーで性能を損なうことなく高速なマルチプレックスリアルタイムRT-PCRを行なえます。ホットスタートと即使用可能なマスターミックス中の独自のPCRバッファーシステムの組み合わせは、最適化実験の必要なしにほとんどのサイクラーで高感度な定量RT-PCRを実現します。2種類のキットが入手できます：蛍光補正のためにROXが必要なサイクラー用QuantiFast Multiplex RT-PCR Kitおよびその他のサイクラー用のQuantiTect Multiplex RT-PCR +R Kit。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。

パフォーマンス

QuantiFast Multiplex RT-PCR KitはRT-PCR時間を最大50％まで短縮できるため、結果がより速く得られます（図 " RT-PCR時間を顕著に短縮"）。従って、サンプル数を大幅に増やしたり、他の実験者と1 台のサイクラーを効率的に共有できます。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増大します（図" 信頼性のある相対定量"）。QuantiFast Multiplex RT-PCR Kitに添付の特化されたマスターミックスにより、迅速なマルチプレックス反応のセットアップができ、singleplex RT-PCRデータに匹敵するマルチプレックスRT-PCR データを提供し、初めての実験でも良好な結果が得られます（図 " triplex とsingleplex RT-PCR で同等の結果を実現"）。

QuantiFast Multiplex RT-PCR Kit を用いると、ターゲット量のわずかな差異を明確に判別できます。本キットは、2 倍希釈しかしていないテンプレートを用いた場合でも、テンプレート中で量が大きく異なるターゲットを正確に定量できます。最高4 種類のターゲットのマルチプレックスリアルタイムPCR が性能を損なうことなく高速で行なえます（図 " 卓越した感度の4-plex RT-PCR"）。

図参照

Significantly reduced RT-PCR times.

Reliable relative quantification.

Comparable results in triplex and singleplex RT-PCR.

Uncompromised sensitivity in 4-plex RT-PCR.

原理

QuantiFast Multiplex RT-PCR Kitsでは最適化実験の必要はなく、標準サイクラーまたは高速サイクラーのどちらでも幅広いダイナミックレンジの高感度かつ迅速な結果が得られます（フローチャート " QIAGEN multiplex kits"）。特別に開発された高速PCR 用バッファーは斬新なQ-Bondを含有し、変性、アニーリングおよびエクステンション時間を顕著に短縮します（図" プライマーの高速なアニーリング"）。

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。QuantiFast Multiplex RT-PCR Buffer のK⁺ およびNH₄⁺ イオン配合比により、プライマーの特異的なアニーリングを促進され、さらにユニークなFactor MP が特異的に結合したプライマーを安定化します（図 " ユニークなPCRバッファー"）。さらに、至適化済み逆転写酵素ミックスにより、わずか20分で効率的なにcDNAを合成できます。また、HotStarTaq Plus DNA Polymeraseは厳密なホットスタートが可能で、非特異的産物の形成を防ぎます。

2x QuantiFast Multiplex RT-PCR Kitの成分
成分	特長	利点
HotStarTaq Plus DNA Polymerase	95℃、5分の活性化	室温での定量PCRのセットアップ
QuantiFast Multiplex RT-PCR Buffer	NH₄⁺ イオンおよび 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PCR結果
	合成添加剤Factor MP	同一チューブで4遺伝子ま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 を含む	PCR反応時間が短縮されるため迅速に結果が得られ、1日あたりの反応数を増やせる
ROX色素^†	Applied Biosystems製装置およびAgilent製装置での蛍光シグナルを補正	ROXが必要なサイクラーで正確な定量。他のリアルタイムサイクラーでの反応を妨害しない
QuantiFast RT Mix	RNAへのアフィニティーが高い逆転写酵素の特殊な配合	複雑な二次構造のRNAでも、わずか20分で転写可能

図参照

QIAGEN multiplex kits.

Fast primer annealing.

Unique buffer promotes stable and efficient primer annealing.

操作手順

QuantiFast Multiplex RT-PCR Kit は反応条件やサイクリング条件の至適化が不要で即使用可能なマスターミックスです。テンプレートRNA、プライマープローブセットをマスターミックスに加えハンドブックのプロトコール通りに操作するだけで、どのリアルタイムサイクラーでも迅速に信頼性の高い結果が得られます。マスターミックス中にROX passive reference dye が入ったキットまたは入っていないキットをお求めいただけますので、実質的にあらゆるリアルタイムサイクラーで使用できます（表を参照）。ROX濃度が至適化されているため、コピー数が少ない場合の検出でも自動データ解析を行なえます。

正しいQuantiFast Multiplex RT-PCR Kitの選択ガイド
ROX 色素	キット	対応するサイクラー
マスターミックスに添加済み	QuantiFast Multiplex RT-PCR Kit	Applied Biosystems 7500以外のApplied Biosystemsの全てのサイクラー
別チューブで添付	QuantiFast Multiplex RT-PCR +R Kit	Applied Biosystems 7500 および Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche、Agilent、その他の会社のサイクラー

アプリケーション

QuantiFast Multiplex RT-PCR Kit はほとんどのリアルタイム用サーマルサイクラーで遺伝子発現解析、その他のアプリケーションに使用可能です。これには、Applied Biosystems およびBio-Rad、Cepheid、Eppendorf、Roche、Agilent 製サイクラーを含みます。Rotor-Gene Q および他のRotor-Gene サイクラーを使用する場合は、これらの高速サイクリング用に開発されたRotor-Gene Multiplex RT-PCR Kitの使用をお勧めします。

裏付けデータと数値

Significantly reduced RT-PCR times.

QuantiFast Multiplex Kits reduce total RT-PCR run time by up to 50% in real-time one-step RT-PCR (40 cycles run; comparison with QuantiTect Multiplex Kits). I: iCycler iQ; L1: LightCycler 480; L2: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500 Fast System; M: Mx3005P.

Specifications

Features	Specifications
Applications	Real-time quantification of RNA targets in a multiplex format
Reaction type	Real-time one-step RT-PCR
Single or multiplex	Multiplex
SYBR Green I or sequence-specific probes	Sequence-specific probes
Real-time or endpoint	Real-time
Thermal cycler	Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
Sample/target type	RNA
With or without ROX	Available with ROX in master mix or with ROX as separate vial

リソース

Download Safety Data Sheets for QIAGEN product components.

For quantitative, multiplex, real-time one-step RT-PCR with fast cycling using sequence-specific probes

FAQ

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

We have successfully tested up to 4 targets per reaction with the QuantiFast Multiplex RT-PCR Kit. A higher degree of multiplexing is achievable only on a limited number of PCR instruments (e.g., LightCycler 2.0). For these rare applications, further optimization of primer–probe concentration, cycling protocol, etc., might be needed.

FAQ ID -2145

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

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

FAQ ID -539

The QuantiFast Buffer in the QuantiFast Multiplex RT-PCR Kit has been further optimized to enable fast multiplex one-step RT-PCR.

FAQ ID -2147

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

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

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

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

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

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

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 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

Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.

FAQ ID -589

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

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

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751

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

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