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

产品

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)

目录编号 / 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

需咨询

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)

目录编号 / 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

需咨询

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"）。

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

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.

Comparable results in triplex and singleplex PCR.

Sensitive duplex PCR and wide dynamic range.

Linear CT values over twofold decreases in template.

规格

特点	规格
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

常见问答

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-1430

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-1976

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-1978

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-1979

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-1981

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-2119

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-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-2371

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-2653

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-2682

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-539

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-589

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-715

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-751

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