Rotor-Gene Multiplex RT-PCR Kit

用于在Rotor-Gene PCR仪上超快速的多重一步法qRT-PCR基因表达分析
  • 在Rotor-Gene PCR仪上获得超快速、可靠的结果
  • 单管内高灵敏检测多个RNA靶序列
  • 表现优越的多重PCR,无需优化
  • 低丰度和高丰度靶基因高效共扩增

Rotor-Gene Multiplex RT-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,可超快速进行基于序列特异性探针的可靠的多重一步法real-time RT-PCR定量分析。通过调整PCR仪,可在单管内同步定量分析多至4个RNA靶基因(如1个参照和3个靶基因)。配合经优化的预混液和具有独特转子设计的Rotor-Gene PCR仪共同使用,表现卓越。为便于使用,预混液应储存在2–8°C。

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Rotor-Gene Multiplex RT-PCR Kit (80)
Trial kit for 80 x 25 µl reactions: 1 ml 2x Rotor-Gene Multiplex RT-PCR Master Mix, 20 µl Rotor-Gene RT Mix, 2 ml RNase-Free Water
204972
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Rotor-Gene Multiplex RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene Multiplex RT-PCR Master Mix, 100 µl Rotor-Gene RT Mix, 2 x 2 ml RNase-Free Water
204974
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The Rotor-Gene Multiplex RT-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.|Fast primer annealing.|QIAGEN multiplex kits.|Unique PCR buffer.|Efficient, sensitive duplex analysis.|Highly efficient 4-plex analysis.|
Duplex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for 28S rRNA (Cyanine 670 dye; data in inset) and PPIA (cyclophilin A, FAM dye). Triplicate reactions were run on the Rotor-Gene 3000 using leukocyte RNA as template (10-fold dilutions from 100 ng to 10 pg). The amplification plots for the duplex reactions (colored) overlapped with those for control singleplex reactions (gray), demonstrating the reliability of the duplex analysis.|[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 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.|Duplex, real-time one-step RT-PCR using in vitro transcripts of [A] HSP90AA1 (109, 107, 105, 103, or 10 copies) and [B] GAPDH (109 copies) was performed (colored curves). For comparison, singleplex RT-PCR was also carried out (gray curves). Duplicate reactions were run on the Rotor-Gene 6000 using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays. Reliable duplex RT-PCR is demonstrated by the evenly spaced curves for HSP90AA1 and overlapping curves for GAPDH. The efficiency of [C] HSP90AA1 amplification was in an optimal range.|4-plex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for the indicated targets. Reactions were run on the Rotor-Gene Q using 100, 10, 1, or 0.1 ng RNA from HeLa cells. The plots of CT value versus log template amount were parallel, indicating all 4 targets were amplified with the same high efficiency. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RPS27A: ribosomal protein S27a; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B-cells.|
Performance
Rotor-Gene Multiplex RT-PCR Kit能够对低至高丰度靶基因可靠进行二重、三重、四重定量检测,可检测低至10 pg的模板及10个拷贝的靶基因(参见"Reliable duplex analysis"和"Efficient, sensitive duplex analysis")。多重分析中不同的靶基因以同样的高效率得以扩增(参见"Highly efficient 4-plex analysis")。因此可实现可靠的相对定量检测,标准品为对照基因。
Principle

在同一反应管中扩增参照和靶基因,减少了手动操作失误,提高了基因定量检测的可靠性。Rotor-Gene Multiplex RT-PCR Kit能够在Rotor-Gene Q实时荧光定量PCR分析仪上可靠的多重定量检测RNA靶基因,无需优化反应条件(参见"QIAGEN multiplex kits")。进行一步法real-time RT-PCR,使逆转录和PCR扩增在同一反应容器中相继进行。因为无需将逆转录产物转移到另一管中进行PCR,所以real-time RT-PCR的流程得以简化,可实现高通量分析。 

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,逆转录酶在15分钟内即可高效的合成cDNA,而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 RT-PCR Kit组分*
组分 特点 优势
HotStarTaq Plus DNA Polymerase 95ºC条件下5分钟激活 室温下建立qPCR反应体系
Rotor-Gene Multiplex RT-PCR Buffer 浓度平衡的NH4+和K+离子 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
Rotor-Gene RT Mix   RNA亲和性高的逆转录酶 RNA逆转录可在15分钟内完成,甚至包括复杂的二级结构
* 都含有dNTP混合液(dATP, dCTP, dGTP, 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RNA和引物探针对、逆转录酶,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RT-PCR分析提供了单独的实验方案。

Applications
Rotor-Gene Multiplex RT-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一步法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。

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 Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
With or without ROX Without ROX dye

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Brochures & Guides
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Kit Handbooks
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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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