使用序列特异性探针进行快速两步法RT-PCR,适用于基因表达分析

  • 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的PCR仪

QuantiFast Probe PCR Kits利用序列特异性探针,可对cDNA进行快速、灵敏的两步法real-time 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。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 PCR Kit。对于其他PCR仪,可选择QuantiFast Probe 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 PCR Kit (4000)
For 4000 x 25 µl reactions: 2 x 25 ml 2x QuantiFast Probe PCR Master Mix (contains ROX dye), 20 ml RNase-Free Water
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QuantiFast Probe 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 PCR times.|Wide dynamic range and high sensitivity.|Faster results without compromising sensitivity.|Sensitive two-step RT-PCR.|Detection over 7 Logs of template.|Specific primer annealing.|Fast primer annealing.|
QuantiFast Probe Kits reduce total PCR run time by up to 60% in real-time two-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.
|The QuantiFast Probe PCR +ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93% in two-step PCR on the Mastercycler ep realplex. Duplicate reactions (25 μl volume) were performed using 10-fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin (a regulatory protein). NTC: no template control.|Reactions were run in duplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan gene expression assay for IL1RN (a cytokine) on the ABI PRISM 7900. The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier AII (which was used according to the standard cycling protocol), providing much lower CT values and transcript quantification from down to 10 pg cDNA.|Reactions were run in triplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a Primer Express designed TaqMan assay for IL8 (a chemokine) on the ABI PRISM 7900. [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 down to 0.01 ng cDNA.|Duplicate two-step RT-PCR reactions were run using a set of primers and FRET probes for b-2 microglobulin from Supplier R on the LightCycler 2.0. The amplification plots for the QuantiFast Kit show the kit's wide dynamic range.|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

QuantiFast Probe PCR Kits可获得高灵敏度结果,优于其它real-time PCR试剂盒(参见"Sensitive two-step RT-PCR on the iCycler iQ"和"Sensitive two-step RT-PCR")。 可缩短60%的PCR运行时间(参见"Significantly reduced PCR times"),更快获得结果,不影响PCR反应效果(参见"Faster results without compromising sensitivity")。可以明显提高您的样品通量,您也可以同其他用户共用PCR仪。

Sensitive two-step RT-PCR on the iCycler iQ
平均CT
人类白血球cDNA (ng) QuantiFast Probe PCR +ROX Vial Kit BV供应商的试剂盒
100 29.13 31.13
10 32.50 34.43
1 36.15 40.00
无模板对照 40.00 40.00
Triplicate reactions were run using a Primer Express designed TaqMan assay for IL8 (a chemokine). The QuantiFast Kit provided lower CT values than the kit from Supplier BV (which was used according to a fast-cycling protocol), allowing transcript quantification from just 1 ng cDNA.

QuantiFast Probe PCR Kits可在大范围内进行准确的定量检测(参见"Wide dynamic range and high sensitivity")。可靠检测稀释倍数高达7个数量级的模板稀释液(参见"Detection over 7 logs of template in two-step RT-PCR on the LightCycler 2.0 "和"Detection over 7 logs of template")。

Detection over 7 logs of template in two-step RT-PCR on the LightCycler 2.0
平均CT
人类白血球cDNA QuantiFast Probe PCR +ROX Vial Kit R供应商的试剂盒
100 ng 11.99 14.56
10 ng 15.54 17.99
1 ng 19.22 22.16
100 pg 22.63 25.14
10 pg 26.43 30.15
1 pg 30.02 34.43
100 fg 33.42 37.10
无模板对照 40 40.00
PCR效率 90% 80.1%
Duplicate reactions were run using a set of primers and FRET probes for b-2 microglobulin from supplier R. The QuantiFast Kit provided lower CT values and higher PCR efficiency that the kit from Supplier R (which was used according to the fast cycling protocol ).
Principle

QuantiFast Probe 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。特制的快速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")。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需要严格的热启动,可避免生成非特异性产物。

2x QuantiFast Probe PCR Kit的成分*
成分 特点 优势
HotStarTaq Plus DNA Polymerase 95ºC加热3分钟活化 在室温进行qPCR反应体系构建
QuantiFast Probe 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
* 也含有dNTP混合液(dATP、dCTP、dGTP和dTTP)。 ROX染料或者在预混液中,或者单独提供。
Procedure

QuantiFast Probe 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。

Choosing the right QuantiFast Probe PCR Kit
ROX染料试剂盒 适用的PCR仪
混合在预混液中 QuantiFast Probe PCR Kit 除Applied Biosystems 7500外的所有Applied Biosystems仪器
单独装在管中 QuantiFast Probe PCR +ROX Vial Kit Applied Biosystems 7500、Agilent、Bio-Rad、Cepheid、QIAGEN、Eppendorf、Roche和其他供应商的PCR仪

为获得最佳的两步法real-time RT-PCR结果,我们推荐应用QuantiTect Reverse Transcription Kit合成cDNA。该试剂盒可在20分钟内快速合成cDNA,同时去除基因组DNA。

QuantiFast Probe Assays是预制的适合全基因组的试剂,使用水解探针法检测;包含在QuantiFast Probe PCR Kit中,可确保在两步法qRT-PCR中获得可靠结果。

Applications

QuantiFast Probe PCR Kits可在任何real-time PCR仪上利用序列特异性探针进行cDNA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 PCR Kit。

Feature
Specifications
Applications Probe-based, real-time PCR, two-step RT-PCR
Reaction type Real-time and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type cDNA, DNA
Single or multiplex Single
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler All real-time cyclers (e.g. Roche LightCycler®, Corbett Rotor-Gene, Applied Biosystems)
With or without ROX Available with ROX in master mix and with ROX as separate vial

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Kit Handbooks
1
For fast, quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
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