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

  • 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

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。

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QuantiFast Multiplex RT-PCR Kit (80)
Trial kit for 80 x 25 µl reactions: 1 ml 2x QuantiFast Multiplex RT-PCR Master Mix (with ROX dye), 20 µl QuantiFast RT Mix, 2 ml RNase-Free Water
204852
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QuantiFast Multiplex RT-PCR Kit (400)
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
204854
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QuantiFast Multiplex RT-PCR +R Kit (80)
Trial kit for 80 x 25 µl reactions: 1 ml 2x QuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 20 µl QuantiFast RT Mix, 45 µl ROX Dye Solution, 2 ml RNase-Free Water
204952
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QuantiFast Multiplex RT-PCR +R Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 100 µl QuantiFast RT Mix, 210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water
204954
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QuantiFast Multiplex RT-PCR +R Kit (2000)
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
204956
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QuantiFast Multiplex RT-PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Significantly reduced RT-PCR times.|Reliable relative quantification.|Comparable results in triplex and singleplex RT-PCR.|Uncompromised sensitivity in 4-plex RT-PCR.|Fast primer annealing.|QIAGEN multiplex kits.|Unique buffer promotes stable and efficient primer annealing.|
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.
|Cell line X was treated with one of two compounds (Y or Z) or left untreated (U). RNA was purified and duplex, real-time one-step RT-PCR was carried out on the Applied Biosystems 7500 Fast System using the QuantiFast Multiplex RT-PCR +R Kit and TaqMan Gene Expression Assays for myogenin and GAPDH. For each treatment, 5 independent experiments were performed. [A] Changes in myogenin expression were detected with high accuracy and reproducibility (green curves). The expression of the housekeeping gene GAPDH was similar in all experiments (blue curves), allowing normalization of myogenin expression levels using the ΔΔCT method of relative quantification. [B] The fold changes in normalized myogenin expression level relative to that in untreated cells were consistent between experiments, as indicated by the small error bars. (Data kindly provided by Angelika Meyer, Novartis Pharma AG, Basel, Switzerland).|Triplex and singleplex, real-time one-step RT-PCR were carried out on the LightCycler 480 using the QuantiFast Multiplex RT-PCR +R Kit and self-designed TaqMan assays for [A] RPS27A (a ribosomal protein), [B] GAPDH (a housekeeping gene), and [C] UBC (a housekeeping gene). The template was Ramos cell line RNA (10 ng, 1 ng, or 0.1 ng), and reactions were run in duplicate. The comparable CT values for triplex PCR (colored curves) and singleplex PCR (gray curves) and [D] the high PCR efficiencies demonstrate the reliability of triplex PCR with the QuantiFast Multiplex RT-PCR +R Kit when analyzing targets of differing abundance.|4-plex, real-time one-step RT-PCR was carried out on the Mx3005P using the QuantiFast Multiplex RT-PCR +R Kit and Primer Express designed TaqMan assays. Duplicate reactions were run using 10 ng RNA from Ramos cells as template. All 4 targets, which varied greatly in abundance, were reproducibly detected.|[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.|QuantiFast Multiplex RT-PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|
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
* 也含有dNTP混合液(dATP、dCTP、dGTP和dTTP)。 †  ROX染料或者在预混液中,或者另外提供。
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。

Feature
Specifications
Applications Real-time quantification of RNA targets in a multiplex format
Reaction type Real-time one-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA
Single or multiplex Multiplex
SYBR Green I or sequence-specific probes Sequence-specific probes
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)
With or without ROX Available with ROX in master mix or with ROX as separate vial

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Kit Handbooks
1
For quantitative, multiplex, real-time one-step RT-PCR with fast cycling using sequence-specific probes
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