使用序列特异性探针进行一步法快速RT-PCR,用于基因表达分析

  • 快速获得结果,节约多达60%的时间
  • 灵敏的检测低拷贝数模板
  • 准确检测各种起始量的模板
  • 优化的即用型预混液用于快速PCR反应
  • 通用的操作流程适用于标准和快速PCR仪

QuantiFast Probe RT-PCR Kits利用序列特异性探针,可对RNA进行快速、灵敏的两步法RT-PCR定量检测。Q-bond技术和优化的预混液使real-time RT-PCR的运行时间缩短,不仅在快速PCR仪上,即使在普通PCR仪上也可快速完成反应。即用型预混液含有热启动酶和独特的PCR缓冲液,确保在任何real-time PCR仪上无需优化即可进行灵敏的定量PCR检测。该试剂盒也提供经优化的RT混合液,10分钟即可高效合成cDNA。提供两种试剂盒规格。对于需要ROX进行荧光校准的PCR仪,可选择QuantiFast Probe RT-PCR Kit。对于其他PCR仪,可选择QuantiFast Probe RT-PCR +ROX Vial Kit。为了便于使用,预混液可保存在2–8°C。

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QuantiFast Probe RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Probe RT-PCR Master Mix (contains ROX dye), 100 µl QuantiFast RT Mix, 2 x 2 ml RNase-Free Water
204454
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QuantiFast Probe RT-PCR Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Probe RT-PCR Master Mix (contains ROX dye), 0.5 ml QuantiFast RT Mix, 20 ml RNase-Free Water
204456
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QuantiFast Probe RT-PCR +ROX Vial Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Probe RT-PCR Master Mix (without ROX dye), 210 µl ROX Dye Solution, 100 µl QuantiFast RT Mix, 2 ml RNase-Free Water
204554
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QuantiFast Probe RT-PCR +ROX Vial Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Probe RT-PCR Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 0.5 ml QuantiFast RT Mix, 20 ml RNase-Free Water
204556
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QuantiFast Probe 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.|Sensitive one-step RT-PCR.|Comparable dynamic range.|Faster results without compromising sensitivity.|Wide dynamic range and high sensitivity.|Specific primer annealing.|Fast primer annealing.|
QuantiFast Probe Kits reduce total RT-PCR run time by up to 60% in real-time one-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.
|Reactions were run in duplicate using 10-fold dilutions of human leukocyte RNA (100 ng to 0.1 ng) and a Primer Express designed TaqMan assay for IL12RBI (a type I transmembrane protein) on the Applied Biosystems 7500 Fast System. [A] The QuantiFast Kit showed greater sensitivity than [B] the kit from Supplier I (which was used according to the standard-cycling protocol), providing lower CT values and transcript quantification from 0.1 ng RNA.|Ubiquitin expression in human leukocytes was analyzed using a Primer Express designed TaqMan assay. Two-step and one-step qPCR reactions were run in duplicate on the ABI PRISM 7900. [A] 10-fold cDNA dilutions (100 ng to 10 pg) were analyzed using the QuantiFast Probe PCR Kit. [B] 10-fold RNA dilutions (100 ng to 10 pg) were analyzed using the QuantiFast Probe RT-PCR Kit.|Reactions were run in triplicate on the ABI PRISM 7900 using 10-fold dilutions of human leukocyte RNA (100 pg to 0.01 pg) and a TaqMan gene expression assay for 28S rRNA. [A] The QuantiFast Kit showed greater sensitivity than [B] the standard-cycling kit from Supplier AII (which was used according to the standard cycling-protocol), providing much lower CT values and quantification from as little as 0.01 pg RNA.|Triplicate reactions were performed using 10-fold dilutions of human leukocyte RNA and a TaqMan assay for β-actin. The QuantiFast Probe RT-PCR Kit provided accurate gene expression analysis from 100 ng down to 1 pg of RNA template.|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in QuantiFast Buffer 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

使用QuantiFast Probe RT-PCR Kits可获得灵敏的结果,比其他real-time RT-PCR试剂盒的性能更加优越(参见"Sensitive One-step RT-PCR")。可节约多达60%的PCR运行时间(参见"Significantly reduced PCR times"),可确保更快获得结果,而RT-PCR的效果不会受到影响(参见"Faster results without compromising sensitivity")。使您可以极大提高样本通量,或通过与其他人员共享PCR仪提高效率。使用QuantiFast Probe RT-PCR Kits不会改变您的操作流程,并适用于标准或快速PCR仪。

QuantiFast Probe RT-PCR Kits可在大范围内进行精确定量检测(参见"Comparable dynamic range")。可靠检测稀释倍数高达6个数量级的模板稀释液(参见"Wide dynamic range and high sensitivity")。

Principle

QuantiFast Probe RT-PCR Kits可在大范围内进行快速、灵敏的定量检测,适用于标准和快速PCR仪。该试剂盒专为各种序列特异性探针设计,包括水解探针(TaqMan探针和其他双标记探针)和FRET探针。经优化的QuantiFast RT Mix可确保在10分钟内合成cDNA。特制的快速RT-PCR缓冲液包含新型Q-Bond成分,可大大缩短变性、退火和延伸时间(参见"Fast primer annealing")。RT-PCR缓冲液中K+和NH4+离子的平衡配比可促进特异性的引物退火,确保高度灵敏和特异的RT-PCR反应(参见"Specific primer annealing")。此外,HotStarTaq Plus DNA Polymerase必须要在95°C加热5分钟才能活化,需要严格的热启动,可避免生成非特异性产物。  

2x QuantiFast Probe 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 Probe 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
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 对需要ROX染料的PCR仪进行校准,不影响PCR反应结果
QuantiFast RT Mix 单独的溶液,需要在反应体系构建时另外加入 在10分钟内快速完成cDNA合成
* 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 Probe RT-PCR Kits包含即用型预混液,无需对反应和扩增条件进行优化。只需按照操作手册进行实验,即可快速获得可靠的结果。提供含有和不含ROX染料的预混液(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 Probe RT-PCR Kit
ROX染料试剂盒 适用的PCR仪
混合在预混液中 QuantiFast Probe RT-PCR Kit 除Applied Biosystems 7500外的所有Applied Biosystems仪器
单独装在管中 QuantiFast Probe RT-PCR +ROX Vial Kit Applied Biosystems 7500、
Mx3000P、Mx3005P、Mx4000和Bio-Rad、Cepheid、Corbett、Eppendorf、和Roche的PCR仪
Applications

QuantiFast Probe RT-PCR Kits利用序列特异性探针,可在任何real-time PCR仪上对目标RNA进行基因表达分析,适用于Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche和Agilent的PCR仪。在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 Probe RT-PCR Kit。

Feature
Specifications
Applications Probe-based, real-time RT-PCR
Reaction type Real-time one-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA
Single or multiplex Single
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX Available with ROX in master mix and with ROX as a separate vial

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Kit Handbooks
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QuantiFast Probe RT-PCR Kit
QuantiFast Probe RT-PCR +ROX Vial Kit For fast, quantitative, real-time, one-step RT-PCR using sequence-specific probes
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