用于在Rotor-Gene PCR仪上使用序列特异性探针超快速进行两步法qRT-PCR

  • 灵敏检测低拷贝数基因
  • 大范围模板量内确保准确的检测
  • Rotor-Gene PCR仪上超快速、可靠的结果
  • 专用的即用型预混液确保快速循环
Rotor-Gene Probe 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,使用序列特异性探针采用定量两步法RT-PCR可超快速、高度灵敏的定量cDNA靶分子。特殊优化的预混液配合独特的Rotor-Gene PCR仪,表现卓越。预混液可贮存在2–8°C,方便使用。

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Rotor-Gene Probe PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x Rotor-Gene Probe PCR Master Mix, 2 x 2 ml RNase-Free Water
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The Rotor-Gene Probe 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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Highly sensitive and reproducible detection.|High precision in real-time PCR.|Reliable detection down to 1 cell.|Wide dynamic range.|Specific primer annealing.|Fast primer annealing.|
Real-time PCR was carried out using two-fold dilutions of human genomic DNA (30 ng to 3.75 ng) and a self-designed TaqMan assay for IL1R2 (interleukin 1 receptor, type II). Reactions were run using either [A] the Rotor-Gene Probe PCR Kit and Rotor-Gene Q or [B] a kit and cycler from Supplier AII (5 replicates per template dilution). The Rotor-Gene system provided lower CT values, greater reproducibility within replicates, and greater resolution of different template dilutions.|Real-time PCR analysis of human genomic DNA was carried out using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. [A] Analysis of two-fold dilutions of DNA (from 30 ng [5000 copies] to 0.06 ng [20 copies]) using a self-designed TaqMan assay for IL1R2 (interleukin 1 receptor, type II); 5 replicates per template dilution. The average difference in CT value between adjacent template dilutions was 1.07 cycles. [B] Analysis of 5 ng DNA using a self-designed TaqMan assay for IL1R2. From 100 replicates, the standard deviation was 0.21. [C] Analysis of 20 ng DNA using a self-designed TaqMan assay for ACTB (actin, beta). From 100 replicates, the standard deviation was 0.29.|Real-time PCR was carried out using fourfold dilutions of human genomic DNA (equivalent to 16, 666 cells down to 1 cell) and a self-designed TaqMan assay for ACTB (actin, beta). Reactions were run in triplicate using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. The Rotor-Gene system provided reliable detection over the entire range of template dilutions, with high reproducibility within each set of triplicates.|Real-time PCR was carried out using ten-fold dilutions of plasmid DNA (109 to 10 copies) and a self-designed TaqMan assay for PPIA (cyclophilin A). Reactions were run in triplicate using the Rotor-Gene Probe PCR Kit and Rotor-Gene Q. The Rotor-Gene system provided reliable detection over the entire range of template dilutions as well as highly reproducible CT values within each set of triplicates.|Cations in the Rotor-Gene Q PCR buffer increase specific primer annealing. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in Rotor-Gene Probe 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.|
Performance

Rotor-Gene Probe PCR Kit非常适合定量PCR分析(参见"Highly sensitive and reproducible detection", "High precision in real-time PCR"),以及在大范围模板量内提供可靠的检测(参见"Reliable detection down to 1 cell", 和 "Wide dynamic range")。

在大范围模板量内可靠检测
拷贝数 平均CT CT差异
1,000,000,000 5.11 3.07
100,000,000 8.18 3.39
10,000,000 11.57 3.04
1,000,000 14.61 3.68
100,000 18.29 3.28
10,000 21.57 3.68
1,000 25.25 3.06
100 28.31 3.43
10 31.74
有效性 0.99
线性 1
Real-time PCR使用10倍稀释的质粒DNA(109至10个拷贝)和为PPIA(cyclophilin A)自己设计的TaqMan分析。
Principle

Rotor-Gene Probe PCR Kit能够在Rotor-Gene Q实时荧光定量PCR分析仪上可靠的定量两步法RT-PCR,无需优化反应或循环条件。该试剂盒专为水解探针(如TaqMan探针)而设计。含有K+和NH4+平衡组合的预混液促进高特异性引物退火,确保高度灵敏和特异性扩增(参见"Specific primer annealing")。新颖的PCR添加剂Q-Bond能够在45分钟内完成循环反应,而不影响表现(参见"Fast primer annealing")。

2x Rotor-Gene Probe 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 在室温下建立qPCR反应体系
Rotor-Gene Probe PCR Buffer 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
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
即用型Rotor-Gene Probe PCR Master Mix无需优化反应和循环条件。只需在预混液中添加模板cDNA、引物和探针,然后设置PCR仪即可开始实验。试剂盒中提供的操作手册有使用说明。

水解探针(如TaqMan探针)与Rotor-Gene Probe PCR Kit配合使用,在Rotor-Gene Q实时荧光定量PCR分析仪上快速、灵敏的定量,只需将引物探针混合物和模板加入预混液即可。

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 Probe 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采用定量两步法RT-PCR可快速定量cDNA靶分子。该试剂盒也与Rotor-Gene 3000和Rotor-Gene 6000兼容。

在Rotor-Gene PCR仪上使用Rotor-Gene Probe RT-PCR Kit,可利用序列特异性探针对RNA靶分子进行超快速的一步法qRT-PCR基因表达分析。

Feature
Specifications
Applications Real-time quantification of genomic DNA or cDNA targets
Description For ultrafast quantitative 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 Single
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 real-time PCR, two-step RT-PCR, and one-step RT-PCR using sequence-specific probes on Rotor-gene cyclers.
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