QuantiFast Multiplex PCR Kits

配列特異的なプローブを用いた迅速なマルチプレックスリアルタイムPCRおよび2ステップ定量RT-PCR

Products

QuantiFast Multiplex 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。
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QuantiFast Multiplex PCR Kit (400)

Cat. No. / ID:  204654

IMPORTANT NOTE: As announced earlier, the production of the QuantiFast kits has been discontinued since mid-2021. Hence, these products will be available only until stocks last.   For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex PCR Master Mix (with ROX dye), 2 x 2 ml RNase-Free Water
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$914.00
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.
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QuantiFast Multiplex PCR +R Kit (2000)

Cat. No. / ID:  204756

IMPORTANT NOTE: As announced earlier, the production of the QuantiFast kits has been discontinued since mid-2021. Hence, these products will be available only until stocks last.   For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water
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$3,551.00
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.

Features

  • 同一チューブ内で再興種類のターゲットを高感度で検出
  • 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PCRを実現
  • 発現量に差異がある複数のターゲットでも信頼性のある定量が可能

Product Details

QuantiFast Multiplex PCR Kit は、マルチプレックスリアルタイムPCRまたは 2ステップRT-PCRにより、同一チューブ中で最高4種類のcDNAまたはゲノムDNAターゲットを高速かつ確実に定量します。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、至適化なしにほとんどのサイクラーで高感度なqRT-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 PCR Kit およびその他のサイクラー用のQuantiFast Multiplex 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。

Performance

QuantiFast Multiplex 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 PCRデータに匹敵するマルチプレックス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 PCRで同等の結果を実現")。本キットを用いると、テンプレート量のわずかな差異を明確に判別できます。QuantiFast Multiplex PCR Kitsは、発現量が大きく異なる2種類のターゲットで、連続2倍希釈したテンプレート量でさえも正確に定量できます(図 " テンプレート量の連続2倍希釈液でも直線性の高いCT")。

QuantiFast Multiplex PCR Kit は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 " PCR時間を顕著に短縮")。わずか10コピーのターゲットを60分で検出できます(図 " 広いダイナミックレンジを持つ高感度なduplex PCR")。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。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において性能を損なうことなく、迅速に結果が得られます(図" 感度を損なわずに増幅")。

See figures

Principle

QuantiFast Multiplex 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 PCR Bufferに入っている画期的な合成添加剤Factor MPとK+およびNH4+のイオン配合比により、プライマーとプローブが核酸テンプレートに効率的かつ安定してアニーリングし、高いPCR効率と感度を実現します(図 " ユニークなPCRバッファー")。さらに、HotStarTaq Plus DNA Polymeraseは厳密なホットスタートを行なえるため、非特異的な産物の形成を抑えます。

2x QuantiFast Multiplex 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 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
*dNTP Mix(dATP、dCTP、dGTPおよびdTTP)も含む。ROX色素はマスターミックス中に含有あるいは別包装の溶液
See figures

Procedure

QuantiFast Multiplex 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 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 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 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

2ステップリアルタイムRT-PCRで最適な結果を得るために、QuantiTect Reverse Transcription Kitを使用してcDNAを合成することをお奨めします。QuantiTect Reverse Transcription Kitは、ゲノムDNAの混入を除去しながらわずか20分で迅速にcDNA を合成します。  

QuantiFast Probe Assaysは加水分解プローブ検出をベースにしたデザイン済みのゲノムワイドなアッセイです。QuantiFast Probe Assaysは、duplexの2ステップリアルタイムRT-PCRで確かな結果が得られるようQuantiFast Multiplex PCR Kit に同梱されています。

Applications

QuantiFast Multiplex PCR Kits はほとんどのリアルタイム用サイクラーでcDNAのマルチプレックス遺伝子発現解析やゲノムDNAターゲットの解析に使用可能です。対応する装置は、Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche社製サイクラーです。Rotor-Gene Q およびその他のRotor-Geneサイクラーに関しては、超高速サイクリング用に特化された Rotor-Gene Multiplex PCR Kitを推奨します。

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsQuantification of cDNA or genomic DNA targets in a multiplex real-time PCR
Thermal cyclerReal-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler® 480, iCycler iQ®, Rotor-Gene)
Real-time or endpointReal-time
Sample/target typecDNA, DNA
SYBR Green I or sequence-specific probesSequence-specific probes
Reaction typeReal-time two-step RT-PCR
Single or multiplexMultiplex
With or without ROXAvailable with ROX in master mix and with ROX as separate vial

Resources

MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
キットハンドブック (1)
For quantitative, multiplex, real-time PCR and two-step RT-PCR with fast cycling using sequence-specific probes
Certificates of Analysis (1)
Kit Handbooks (1)
For quantitative, multiplex, real-time PCR and two-step RT-PCR with fast cycling using sequence-specific probes

FAQ

How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

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
Do limiting primer concentrations need to be determined when using QuantiFast Multiplex PCR Kits?

No, that is not necessary. Simply use the primer concentrations specified in the protocols in the handbook supplied with your QuantiFast Multiplex PCR Kit.

 

 

FAQ ID -1976
I would like to order the QuantiFast Probe Assays without master mix. Is this possible?

QuantiFast Probe Assays are only sold in combination with optimized real-time RT-PCR master mixes and cannot be sold as stand-alone assays.

 

FAQ ID -2360
Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

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.

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
Can the QuantiFast Multiplex PCR Kits be used on Roche LightCycler systems with TaqMan® probes?
  • LightCycler 1.5: We do not recommend performing multiplex, real-time PCR using TaqMan® probes on this LightCycler system due to the limitations of its optical detection system
  • LightCycler 2.0: Information on using the QuantiFast Multiplex PCR +R Kit with the LightCycler 2.0 system is provided in the QuantiFast Multiplex PCR Handbook.

See trademarks

FAQ ID -1979
Why are the QuantiTect and QuantiFast Multiplex PCR Kits limited to triplex real-time PCR on some cyclers?

The presence of ROX as passive reference dye in the Master Mix of the QuantiTect Multiplex PCR Kit and the QuantiFast Mutliplex PCR Kit limits the use of these kits to triplex PCR on 4-channel real-time cyclers. This is because the ROX dye occupies the channel for detecting probes labeled with ROX, Texas Red, or other equivalent dyes.

However, 4-plex PCR is possible on on instruments equipped with at least 5 channels. Alternatively, if you have a 4-channel real-time cycler that does not use ROX passive reference dye (e.g., iCycler iQ, Rotor-Gene 3000, Mx4000, Mx3000P, Smart Cycler II, LightCycler 2.0), you can use the QuantiTect Multiplex PCR NoROX Kit or the QuantiFast Multiplex PCR +R Kit to perform 4-plex PCR.

Please see table “Real-Time, Multiplex PCR on a Wide Range of Real-Time Cyclers” for compabilities of the QuantiTect Multiplex kits with different real-time cyclers.

FAQ ID -715
Why do replicates in real-time PCR have different plateau heights?

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
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

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
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

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
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

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
What is the threshold cycle or Ct value?
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
What do I do if no fluorescent signal is detected in a real-time PCR assay?

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
How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling?

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
How do I select appropriate reporter and quencher combinations for multiplex PCR?

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
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

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
Why does my realtime PCR assay quality decrease over time?
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
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. 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
How do I ensure reliable results for High Resolution Melting (HRM) assays?

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 CT values are below 30 and differ no more than 3 CT 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
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

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
Is the QuantiFast Multiplex PCR +R Kit compatible with the ViiA7 cycler from Applied Biosystems?
We got comparable results on ABI 7500 and the ViiA7 using the QuantiFast Multiplex PCR +R Kit master mix with low ROX concentration (ROX added to NoROX master mix) and the protocol for the ABI 7500.
FAQ ID -2653
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

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
Are QuantiFast Multiplex PCR Kits available in trial sizes?

Yes, we offer trial-size kits for 80 x 25 µl reactions; cat. no. 204652 for the QuantiFast Multiplex PCR Kit, and cat. no. 204752 for the QuantiFast Multiplex PCR +R Kit.

 

FAQ ID -1981
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

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 (Tm) 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 Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096
Can 4-plex, real-time PCR be performed on the ABI PRISM 7000, 7700, or 7900 using the QuantiFast Multiplex PCR Kit?

No, the QuantiFast Multiplex PCR Kit cannot be used for 4plex real-time PCR on the ABI PRISM 7000, 7700, or 7900 instruments. Due to hardware limitations, the maximum capacity of these real-time cyclers is triplex PCR.

 

FAQ ID -1978