QuantiFast Multiplex PCR Kits

使用序列特异性探针进行快速的多重两步法RT-PCR，适用于基因表达分析

Products

QuantiFast Multiplex 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 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

Inquire

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.

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

Inquire

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.

特点

灵敏检测单管中的多个目标
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PCR
准确区分起始量相近的模板

产品详情

应用QuantiFast Multiplex PCR Kit可在单管内通过多重两步法real-time PCR对多至4个cDNA进行快速、可靠的定量检测。Q-Bond和优化的预混液促进快速多重real-time 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 PCR Kit适用于需要ROX染料进行荧光信号校准的PCR仪，QuantiFast Multiplex 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。

绩效

随QuantiFast Multiplex PCR Kit提供的特制的预混液可快速构建多重反应，得到的多重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 PCR"）。该试剂盒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（参见" Linear C_T values over twofold decreases in template"）。

QuantiFast Multiplex PCR Kits可节省多达50%的PCR反应时间，让您更快获得结果（参见" Significantly reduced PCR times"）。只需60分钟，即可检测到低至10个拷贝的靶基因（参见" Sensitive duplex PCR and a wide dynamic range"）。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。该试剂盒可对多达4个靶基因进行快速、多重real-time PCR，且不会影响PCR效果（参见" Uncompromised sensitivity"）。

查看图表

Comparable results in triplex and singleplex PCR.

Linear CT values over twofold decreases in template.

Significantly reduced PCR times.

Sensitive duplex PCR and wide dynamic range.

Uncompromised sensitivity.

原理

QuantiFast Multiplex PCR Kits可在标准或快速PCR仪上快速、灵敏的获得反应结果，无需优化（参见" QIAGEN multiplex kits"）。特制的快速PCR缓冲液含有新型Q-Bond成分，可明显缩短变性、退火和延伸的时间（参见" Fast primer annealing"）。

在同一个反应中同时扩增对照和靶基因，而非分开反应，减少了操作误差，提高了检测的可靠性。QuantiFast Multiplex PCR Buffer 含有平衡配比的K⁺和NH₄⁺以及独特的MP因子，二者可促进引物、探针与核酸模板的稳定高效结合，确保高效的PCR反应（参见" Unique PCR buffer"）此外，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 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 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	在单管中可靠的对至多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	对需要ROX染料的PCR仪进行校准，不影响PCR反应结果

查看图表

Fast primer annealing.

Unique PCR buffer.

程序

QuantiFast Multiplex PCR Kit含有即用型预混液，无需优化反应及循环条件。只需将cDNA和引物探针对加入预混液中，按照操作手册进行实验，即可快速获得可靠的结果，适用于所有real-time PCR仪。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，可用于所有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 PCR Kit
ROX染料	试剂盒	适用的PCR仪
混合在预混液中	QuantiFast Multiplex PCR Kit	除Applied Biosystems 7500外的所有Applied Biosystems仪器
单独装在管中	QuantiFast Multiplex PCR +R Kit	Applied Biosystems 7500、Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche、Agilent和其他供应商的PCR仪

为获得最佳的两步法real-time RT-PCR结果，我们建议使用QuantiTect Reverse Transcription Kit合成cDNA。该试剂盒可在20分钟进行快速cDNA合成，并去除基因组DNA污染。

QuantiFast Probe Assays利用序列特异性水解探针检测技术，是适合全基因组的预制试剂。随QuantiFast Multiplex PCR Kit提供，可确保在双染两步法real-time RT-PCR反应中获得可靠结果。

应用

QuantiFast Multiplex PCR Kit可用在各种real-time PCR仪上对cDNA进行多重基因表达分析，适用仪器包括Agilent、Applied Biosystems、Bio-Rad、Cepheid、Eppendorf和Roche的PCR仪。在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 PCR Kit。

辅助数据和图表

Significantly reduced PCR times.

QuantiFast Multiplex Kits reduce total PCR run time by up to 50% in real-time two-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	Quantification of cDNA or genomic DNA targets in a multiplex real-time PCR
Thermal cycler	Real-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 endpoint	Real-time
Sample/target type	cDNA, DNA
SYBR Green I or sequence-specific probes	Sequence-specific probes
Reaction type	Real-time two-step RT-PCR
Single or multiplex	Multiplex
With or without ROX	Available with ROX in master mix and with ROX as separate vial

资源

Download Safety Data Sheets for QIAGEN product components.

For quantitative, multiplex, real-time PCR and two-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

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

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

For one-step RT-PCR with singleplex detection: QuantiFast Probe RT-PCR Plus Kit
For one-step RT-PCR with duplex detection: QuantiFast Probe RT-PCR Plus Kit
For two-step RT-PCR with singleplex detection: QuantiFast Probe PCR Kit
For two-step RT-PCR with duplex detection: QuantiFast Multiplex PCR Kit

FAQ ID -2360

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

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

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

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

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 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

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

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

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

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, 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

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

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