QuantiFast Multiplex RT-PCR Kits

使用序列特异性探针进行快速的多重一步法RT-PCR，适用于基因表达分析

Products

QuantiFast Multiplex RT-PCR Kits 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

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.

Features

灵敏检测单管中的多个目标RNA
快速获得结果，可节省多达50%的时间
无需优化，即可成功进行多重RT-PCR
准确区分起始量相近的模板
可检测同一反应管内的对照基因和至多3个靶基因

Product Details

应用QuantiFast Multiplex RT-PCR Kits可在单管内通过多重一步法real-time RT-PCR对多至4个RNA进行快速、可靠的定量检测。特制的逆转录酶混合液可快速、高效合成cDNA。Q-Bond和优化的预混液促进快速多重real-time RT-PCR的进行，适用于快速或标准PCR仪。即用型预混液中的热启动酶和独特的PCR缓冲液可确保在所有real-time PCR仪上进行灵敏的qPCR，无需优化。有两种规格的试剂盒：QuantiFast Multiplex RT-PCR Kit适用于需要ROX染料进行荧光信号校准的PCR仪，QuantiFast Multiplex RT-PCR +R Kit适用于其他所有PCR仪。为便于使用，预混液可保存在2–8°C。

Performance

QuantiFast Multiplex RT-PCR Kits可节省高达50%的RT-PCR运行时间，让您更快获得结果（参见"Significantly reduced RT-PCR times"）。您可极大地提高样本通量或与其它使用者有效地共享PCR仪。在同一个反应中同时扩增对照基因和靶基因，而非分开反应，减少了操作误差，提高了基因定量检测的可靠性（参见"Reliable relative quantification"）。QuantiFast Multiplex RT-PCR Kits提供的特制预混液可快速构建多重反应，通常初次使用即可得到成功的结果，且多重PCR反应结果可与单重PCR反应结果相媲美（参见"Comparable results in triplex and singleplex RT-PCR"）。

QuantiFast Multiplex RT-PCR Kits可清楚区分模板量的细微差别。即使模板量仅有两倍的差别，该试剂盒也能精确定量丰度差别较大的2个靶基因。该试剂盒可对多达4个靶基因进行快速多重real-time RT-PCR检测，且不会影响PCR结果（参见"Uncompromised sensitivity in 4-plex RT-PCR"）。

Principle

QuantiFast Multiplex RT-PCR Kits在标准和快速PCR仪上快速、灵敏的进行检测，无需优化（参见"QIAGEN multiplex kits"）。特制的快速PCR缓冲液中含有新型成分Q-Bond，可大大缩短变性、退火和延伸所需时间（参见"Fast primer annealing"）。

在同一个反应中同时扩增对照基因和靶基因，而非分开反应，减少了操作误差，提高了基因定量检测的可靠性。QuantiFast Multiplex RT-PCR Buffer含有平衡的K⁺和NH₄⁺配比，促进了引物的特异性退火，同时独特的MP因子可稳定特异性结合的引物（参见"Unique PCR buffer"）。此外，经优化的逆转录酶混合液确保cDNA合成在20分钟内完成。HotStarTaq Plus DNA Polymerase要求严谨的热启动，可阻止非特异性产物的形成。

2x QuantiFast Multiplex RT-PCR Kit的成分
成分	特点	优势
HotStarTaq Plus DNA Polymerase	95ºC加热5分钟活化	在室温进行qPCR反应体系构建
QuantiFast Multiplex RT-PCR Buffer	平衡的NH₄⁺和K⁺离子配比	引物探针的特异性结合确保获得可靠结果
	合成的MP因子	在单管中对至多4个基因进行多重分析
	独特的Q-Bond添加剂	PCR运行时间缩短，更快获得结果，一天内可完成更多PCR反应
ROX染料^†	对Applied Biosystems和Agilent PCR仪进行荧光信号的校准	在需要ROX染料的PCR仪上进行准确定量，不影响PCR反应结果
QuantiFast RT Mix	特制的逆转录酶混合液，对RNA具有高度亲和性	RNA的逆转录可在20分钟内完成，包括复杂的二级结构

Procedure

QuantiFast Multiplex RT-PCR Kits提供即用型的预混液，无需优化反应及循环条件。只需在预混液中加入模板RNA和引物-探针对，即可按照操作手册中的实验方案，在任何real-time PCR仪上进行快速、可靠的检测。试剂盒提供两种形式：预混液中含有或不含荧光校正用的ROX染料，因此适用于任何real-time PCR仪（参见下表）。由于ROX浓度经优化，即使是低拷贝数也可通过数据自动分析进行检测。

选择合适的QuantiFast Multiplex RT-PCR Kit
ROX染料	试剂盒	适用的PCR仪
混合在预混液中	QuantiFast Multiplex RT-PCR Kit	除Applied Biosystems 7500外的所有Applied Biosystems仪器
单独装在管中	QuantiFast Multiplex RT-PCR +R Kit	Applied Biosystems 7500、 Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche、Agilent和其他供应商的PCR仪

Applications

QuantiFast Multiplex RT-PCR Kits可用于在各种real-time PCR仪上进行基因表达分析，包括Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche和Agilent的PCR仪。Rotor-Gene Q实时荧光定量PCR分析仪或其他Rotor-Gene PCR仪上使用时，我们推荐使用专为快速PCR研发的Rotor-Gene Multiplex RT-PCR Kit。

Supporting data and figures

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.

Uncompromised sensitivity in 4-plex RT-PCR.

Unique buffer promotes stable and efficient primer annealing.

Comparable results in triplex and singleplex RT-PCR.

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

Resources

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