用于在Rotor-Gene PCR仪上超快速的两步法多重qRT-PCR基因表达分析

  • 在Rotor-Gene PCR仪上获得超快速、可靠的结果
  • 单管内灵敏检测多个靶序列
  • 表现优越的多重PCR,无需优化
  • 准确检测靶基因的微小差异

Rotor-Gene Multiplex PCR Kit专用于Rotor-Gene Q实时荧光定量PCR分析仪和其他Rotor-Gene PCR仪,进行超快速、高度可靠的多重定量两步法RT-PCR,使用序列特异性探针。通过调整热循环仪,可在同一反应中同时定量分析多达4个cDNA靶基因(如1个对照基因和3个靶基因)。特殊优化的预混液配合独特的Rotor-Gene PCR仪,表现卓越。为方便使用,预混液可储存在2–8°C。

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Rotor-Gene Multiplex PCR Kit (80)
Trial kit for 80 x 25 µl reactions: 1 ml 2x Rotor-Gene Multiplex PCR Master Mix, 2 ml RNase-Free Water
204772
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Rotor-Gene Multiplex PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene Multiplex PCR Master Mix, 2 x 2 ml RNase-Free Water
204774
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The Rotor-Gene Multiplex PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Reliable duplex analysis.|Reliable multiplex analysis.|Fast primer annealing.|QIAGEN multiplex kits.|Unique PCR buffer.|
Duplex, real-time two-step RT-PCR was carried out on the Rotor-Gene Q using the Rotor-Gene Multiplex PCR Kit and self-designed TaqMan assays for [A] IL8 (interleukin 8) and [B] ACTB (β-actin). Analysis of tenfold dilutions of leukocyte cDNA template from 100 ng to 1 pg provided high PCR efficiencies of around 95%. [C] The CT values were comparable with those achieved in control singleplex reactions, demonstrating the reliability of the duplex assay.|Tenfold dilutions of human leukocyte cDNA (10 ng to 10 pg) were used as template in 4-plex, real-time PCR. Reactions were run in triplicate using either the Rotor-Gene Q and Rotor-Gene Multiplex PCR Kit or an instrument and kit from Supplier S. [A] Targets amplified, and reporter dyes of corresponding TaqMan probes. [B] CT values obtained for all 4 targets (instrument and kit from Supplier S did not successfully detect IFNG; N.D.). Lower CT values on the Rotor-Gene Q demonstrate detection with greater sensitivity. [C] Amplification plots for TNF using the Rotor-Gene Multiplex PCR Kit. [D] Amplification plots for TNF using a kit from Supplier S. [E] Amplification plots for IFNG using the Rotor-Gene Multiplex PCR Kit. [F] Amplification plots for IFNG using a kit from Supplier S.|[A] Q-Bond in Rotor-Gene Multiplex PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer 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 primer annealing time.|Rotor-Gene Multiplex 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
使用Rotor-Gene Multiplex RT-PCR Kit,多达4个cDNA靶基因可在单管内同步快速定量分析,提高通量的同时,节约珍贵的样本(参见 "Reliable multiplex analysis" and "Reliable duplex analysis")。多重分析中不同表达水平的靶基因在同一个反应管中以同样的效率扩增,可确保可靠的相对定量分析。
Principle
扩增参照和靶基因在同一个反应管中,减少操作误差,提高了基因定量分析的可靠性。Rotor-Gene Multiplex RT-PCR Kit确保在Rotor-Gene Q实时荧光定量PCR分析仪上进行可靠的多重cDNA靶基因定量分析,无需优化反应和循环条件(参见"QIAGEN multiplex kits")。 平衡的K+和NH4+离子组合确保高度特异性,促进特异性引物退火,PCR特异性高、灵敏度好。一种新颖的PCR添加剂合成的Factor MP专用于多重PCR应用,能够以同样的效率扩增在同一反应中不同的扩增子(参见 "Unique PCR buffer")。

一种新颖的PCR添加剂Q-Bond可缩短扩增时间,确保快速扩增的同时不影响表现(参见"Fast primer annealing")。 此外,高度严谨的热启动酶HotStarTaq Plus DNA Polymerase在95ºC条件下只需5分钟即可快速激活。

2x Rotor-Gene Multiplex PCR Kit*的组分
组分 特性 优势
HotStarTaq Plus DNA聚合酶 95ºC条件下5分钟激活 室温下建立qPCR反应体系
Rotor-Gene Multiplex PCR缓冲液 NH4+和K+平衡组合预混液 特异性引物退火,确保可靠的qPCR结果
合成的Factor MP 在同一反应管中可靠的进行多达4个基因的多重分析
独特的Q-Bond添加剂 更快速的PCR循环,快速获得结果,一天可进行更多的反应
*  包含dNTP混合液(dATP、dCTP、dGTP和dTTP)。
Procedure

应用即用型预混液无需优化反应和循环条件。只需在预混液中添加模板cDNA和引物探针对,然后设置PCR仪即可开始实验。试剂盒中提供的操作手册中列出了推荐染料,并为多重qPCR分析提供了单独的实验方案。

为了两步法定量RT-PCR获得更加优化的结果,推荐使用QuantiTect Reverse Transcription Kit合成cDNA。该试剂盒只需20分钟即可快速合成cDNA,并可完全去除基因组DNA的污染。

Applications

Rotor-Gene Multiplex PCR Kit专用于Rotor-Gene Q实时荧光定量PCR分析仪,使用序列特异性探针进行快速的两步法RT-PCR分析。该试剂盒也与Rotor-Gene 3000和Rotor-Gene 6000兼容。多达4个cDNA靶基因可在单管内同步快速定量分析,提高通量的同时,节约珍贵的样本。

Rotor-Gene Multiplex RT-PCR Kit用于Rotor-Gene PCR仪上,使用序列特异性探针对RNA靶分子进行超快速的一步法qRT-PCR多重分析。

Feature
Specifications
Applications Real-time quantification of genomic DNA or cDNA targets in a multiplex format
Description For ultrafast quantitative multiplex real-time PCR and two-step RT-PCR using sequence-specific probes
Reaction type Real-time PCR and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type DNA, cDNA
Single or multiplex Multiplex
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
With or without ROX Without ROX dye

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For fast multiplex real-time PCR, two-step RT-PCR, and one-step RT-PCR using sequence-specific probes on Rotor-Gene cyclers
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