QuantiFast Multiplex RT-PCR Kits

For fast, multiplex, one-step qRT-PCR using sequence-specific probes for gene expression analysis

  • Sensitive detection of multiple RNA targets in 1 well
  • Faster results with time savings of up to 50%
  • Successful multiplex RT-PCR without the need for optimization
  • Precise discrimination of small differences in target amount
  • Detection of reference gene and up to 3 targets in the same tube

QuantiFast Multiplex RT-PCR Kits enable fast and reliable quantification of up to 4 RNA targets in a single tube by multiplex, real-time one-step RT-PCR. A special blend of reverse transcriptases delivers fast and efficient cDNA synthesis. Q-bond technology and an optimized master mix promote fast, multiplex real-time RT-PCR, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. Two kit formats are available: the QuantiFast Multiplex RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex RT-PCR +R Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.

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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
询价
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
询价
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
询价

QuantiFast Multiplex RT-PCR Kits适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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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 reduce RT-PCR run times by up to 50%, allowing you to get results significantly faster (see figure "Significantly reduced RT-PCR times"). You can also greatly increase your sample throughput or efficiently share a cycler with other users. Amplifying control and target genes in the same reaction, instead of in separate reactions, increases the reliability of gene quantification by minimizing handling errors (see figure "Reliable relative quantification"). The special master mix supplied with QuantiFast Multiplex RT-PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex RT-PCR data that are comparable with singleplex RT-PCR data (see figure "Comparable results in triplex and singleplex RT-PCR").

QuantiFast Multiplex RT-PCR Kits can clearly distinguish between small differences in the amount of template. Even with two-fold differences in template amount, the kits provide accurate quantification of targets of widely differing abundance. Fast results in multiplex, real-time RT-PCR of up to 4 targets are achieved without compromising performance (see figure "Uncompromised sensitivity in 4-plex RT-PCR").

Principle

QuantiFast Multiplex RT-PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization (see flowchart "QIAGEN multiplex kits"). The specially developed fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing").

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiFast Multiplex RT-PCR Buffer includes a balanced combination of K+ and NH4+ ions to promote specific primer annealing, while unique Factor MP stabilizes specifically bound primers (see figure "Unique PCR buffer"). In addition, an optimized mix of reverse transcriptases provides efficient cDNA synthesis in just 20 minutes, while HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Components of 2x QuantiFast Multiplex RT-PCR Kit
Component Features Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast Multiplex RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
Synthetic Factor MP Reliable multiplexing analysis of up to 4 genes in the same tube
Unique Q-Bond additive Faster PCR run times, enabling faster results and more reactions per day
ROX dye Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler
QuantiFast RT Mix Special blend of reverse transcriptases with high affinity for RNA RNA can be transcribed in just 20 minutes, even through complex secondary structures
*  Also contains a dNTP mix (dATP, dCTP, dGTP, and dTTP).
 ROX dye is either present in the master mix or as a separate solution.
Procedure

QuantiFast Multiplex RT-PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template RNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table).  Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

Choosing the right QuantiFast Multiplex RT-PCR Kit
ROX dyeKit Compatible cyclers
Supplied in master mix QuantiFast Multiplex RT-PCR Kit All cyclers from Applied Biosystems except Applied Biosystems 7500
Supplied in separate tube QuantiFast Multiplex RT-PCR +R Kit Applied Biosystems 7500 and cyclers from
Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers
Applications

QuantiFast Multiplex RT-PCR Kits can be used for multiplex gene expression analysis of RNA targets on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Multiplex RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

特点
参数
应用 Real-time quantification of RNA targets in a multiplex format
反应类型 Real-time one-step RT-PCR
real-time或终点法PCR Real-time
样本/目标类型 RNA
单一或多重 Multiplex
SYBR Green I或序列特异性探针 Sequence-specific probes
热循环仪 Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
有/无ROX Available with ROX in master mix or with ROX as separate vial

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For quantitative, multiplex, real-time one-step RT-PCR with fast cycling using sequence-specific probes
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Significantly reduced PCR times
RT-PCR时间极大缩短。
使用QuantiFast Multiplex Kit进行一步法real-time RT-PCR,可缩短PCR总运行时间达50%(40个循环运行;与QuantiTect Multiplex Kit相比较)。I:iCycler iQ;L1:LightCycler 480;L2:LightCycler 2.0;A1:ABI PRISM 7900;A2:Applied Biosystems 7500 Fast System;M:Mx3005P。
Reliable relative quantification
可靠的相对定量。
细胞系X采用两种化合物之一 (YZ) 处理或者不处理 (U)。纯化RNA,在Applied Biosystems 7500 Fast System上使用QuantiFast Multiplex RT-PCR +R Kit和针对肌细胞生成素和GAPDH的TaqMan Gene Expression Assay进行一步法双重real-time RT-PCR。针对每次处理,进行5次独立实验。[A] 检测肌细胞生成素表达的变化,检测的准确度和重复性较高 (绿色曲线)。在所有实验中,管家基因GAPDH的表达相当 (蓝色曲线),可利用ΔΔCT相对定量方法对肌细胞生成素的表达水平进行标准化。[B] 在不同实验之间,相对于未处理细胞,标准化的肌细胞生成素表达水平的倍数变化保持稳定,如图中较小的误差条所示。(数据由瑞士巴塞尔Novartis Pharma AG的Angelika Meyer提供)。
Comparable results in triplex and singleplex RT-PCR on the LightCycler 480
三重和单重RT-PCR的结果具有可比性。
在LightCycler 480上使用QuantiFast Multiplex RT-PCR +R Kit和采用Primer Express设计的针对 [A] RPS27A (一种核糖体蛋白)、[B] GAPDH (管家基因) 和 [C] UBC (管家基因) 的TaqMan Assay进行三重和单重real-time RT-PCR。模板为HeLa细胞cDNA (10 ng、1 ng或0.1 ng),进行两次重复反应。三重PCR (彩色曲线) 和单重PCR (灰色曲线) 的比较CT值证明了在分析不同丰度的靶点时,使用QuantiFast Multiplex RT-PCR +R Kit进行三重PCR的可靠性。
Uncompromised sensitivity in 4-plex RT-PCR on the Mx3005P.
四重RT-PCR具有高灵敏性。
在Mx3005P上使用QuantiFast Multiplex RT-PCR +R Kit和采用Primer Express设计的TaqMan Assay进行4重一步法real-time RT-PCR。使用10 ng Ramos细胞RNA为模板,进行两次重复反应。以可重复的方法检测所有4个丰度差异极大的靶点。
Fast primer annealing with Q-Bond
退火时引物快速结合。
[A] QuantiFast Buffer中的Q-Bond能使DNA聚合酶和引物结合为单一复合物,将退火时间缩短至几秒。此外,独特的缓冲液组分可支持DNA的熔解,缩短变性和延伸时间。[B] 若无Q-Bond,引物和聚合酶会相继与模板结合,退火时间较长。
Successful Multiplex PCR-Based Genotypic Analysis without the Need for Optimization
QIAGEN多重试剂盒。
QuantiFast Multiplex RT-PCR Kit提供了简单的多重定量real-time PCR步骤。与当前的方法相比,即便是进行复杂的分析(如目的基因的拷贝数明显少于对照基因的分析),该试剂盒亦无需优化引物、Mg2+Taq DNA聚合酶的浓度,甚至是困难模板(例如:靶基因拷贝数大大少于参考基因)。
Unique Type-it Microsatellite PCR Buffer Promotes Stable and Efficient Annealing
独特的缓冲液能够促进稳定、高效的退火。
[A] NH4+防止引物与模板的非特异性结合。[B] MP合成因子是创新的PCR添加剂,能够提高模板处局部的引物浓度,与K+和其他离子一起,MP合成因子能够特异性稳定结合的引物,使HotStarTaq Plus DNA Polymerase作用下的引物延伸更加高效。