QuantiFast Multiplex PCR Kits
使用序列特异性探针进行快速的多重两步法RT-PCR,适用于基因表达分析
- 灵敏检测单管中的多个目标
- 快速获得结果,可节省多达50%的时间
- 无需优化,即可成功进行多重PCR
- 准确区分起始量相近的模板
应用QuantiFast Multiplex PCR Kit可在单管内通过多重两步法real-time PCR对多至4个cDNA进行快速、可靠的定量检测。Q-Bond和优化的预混液促进快速多重real-time PCR的进行,适用于快速或标准PCR仪。即用型预混液中的热启动酶和独特的PCR缓冲液可确保在所有real-time PCR仪上进行灵敏的qPCR,无需优化。有两种规格的试剂盒:QuantiFast Multiplex PCR Kit适用于需要ROX染料进行荧光信号校准的PCR仪,QuantiFast Multiplex PCR +R Kit适用于其他所有PCR仪。为便于使用,预混液可保存在2–8°C。
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QuantiFast Multiplex PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex PCR Master Mix (with ROX dye), 2 x 2 ml RNase-Free Water
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204654
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QuantiFast Multiplex PCR Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex PCR Master Mix (with ROX dye), 20 ml RNase-Free Water
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204656
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QuantiFast Multiplex PCR +R Kit (80)
Trial kit for 80 x 25 µl reactions: 1 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 45 µl ROX Dye Solution, 2 ml RNase-Free Water
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204752
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QuantiFast Multiplex PCR +R Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water
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204754
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QuantiFast Multiplex PCR +R Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water
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204756
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QuantiFast Multiplex PCR +R Kit (4000)
For 4000 x 25 µl reactions: 2 x 25 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 2 x 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water
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204757
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QuantiFast Multiplex PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
PCR时间极大缩短。|灵敏的PCR,动态范围宽泛。|三重PCR和单重PCR的结果相当。|高度灵敏性。|退火时引物快速结合。|QIAGEN多重试剂盒。|独特的PCR缓冲液。|模板量降低,CT值仍呈线性。|
使用QuantiFast Multiplex Kit进行两步法real-time RT-PCR,可缩短PCR总运行时间达50%(40个循环运行;与QuantiTect Multiplex Kit相比较)。I:iCycler iQ;L1:LightCycler 480;L2:LightCycler 2.0;A1:ABI PRISM 7900;A2:Applied Biosystems 7500 Fast System;M:Mx3005P。|使用DNA模板混合物在Applied Biosystems 7500 Fast System上进行两次重复反应,混合物中包含108个拷贝的β-actin(数据在插图中显示)和106至10个拷贝的RPS27A (核糖体蛋白)。[A] QuantiFast Multiplex PCR +R Kit的灵敏度明显高于[B] Supplier AII的双重PCR试剂盒,使得在快速循环模式下运行的循环仪能够检测低至10拷贝的靶点,并定量超过6 log稀释范围的模板。|在LightCycler 480上使用QuantiFast Multiplex PCR +R Kit和采用Primer Express设计的针对 [A] RPS27A (一种核糖体蛋白)、[B] GAPDH (管家基因) 和 [C] UBC (管家基因) 的TaqMan Assay进行三重和单重PCR。模板为HeLa细胞cDNA (10 ng、1 ng或0.1 ng),进行两次重复反应。三重PCR (彩色曲线) 和单重PCR (灰色曲线) 的比较CT值证明了在分析不同丰度的靶点时,使用QuantiFast Multiplex PCR +R Kit进行三重PCR的可靠性。|使用QuantiFast Multiplex PCR +R Kit和采用Primer Express设计的TaqMan Assay在Applied Biosystems 7500 Fast System上进行4重real-time PCR。使用10 ng Ramos细胞系cDNA进行三次重复反应。|[A] QuantiFast Buffer中的Q-Bond能使DNA聚合酶和引物结合为单一复合物,将退火时间缩短至几秒。此外,独特的缓冲液组分可支持DNA的熔解,缩短变性和延伸时间。[B] 若无Q-Bond,引物和聚合酶会相继与模板结合,退火时间较长。|QuantiFast Multiplex PCR Kit提供了简单的多重定量real-time PCR步骤。与当前的方法相比,即便是进行复杂的分析(如目的基因的拷贝数明显少于对照基因的分析),该试剂盒亦无需优化引物、Mg2+和Taq DNA聚合酶的浓度。|[A] NH4+防止引物与模板的非特异性结合。[B] MP合成因子是创新的PCR添加剂,能够提高模板处局部的引物浓度,与K+和其他离子一起,MP合成因子能够特异性稳定结合的引物,使HotStarTaq Plus DNA Polymerase作用下的引物延伸更加高效。|在Applied Biosystems 7500 Fast System上使用QuantiFast Multiplex PCR +R Kit及针对t (8;14) 染色体易位和GAPDH的分析进行双重和单重PCR。使用Ramos细胞系的基因组DNA为模板 (10 ng至0.625 ng的两倍稀释),进行四重反应。在 [A] 单重和 [B] 双重反应中,CT值随着模板稀释度的降低呈线性升高,上升幅度为1个CT值,说明该试剂盒能够精确区分模板量的微小差异。|
原理
QuantiFast Multiplex PCR Kits可在标准或快速PCR仪上快速、灵敏的获得反应结果,无需优化(参见"QIAGEN multiplex kits")。特制的快速PCR缓冲液含有新型Q-Bond成分,可明显缩短变性、退火和延伸的时间(参见"Fast primer annealing")。
在同一个反应中同时扩增对照和靶基因,而非分开反应,减少了操作误差,提高了检测的可靠性。QuantiFast Multiplex PCR Buffer 含有平衡配比的K+和NH4+以及独特的MP因子,二者可促进引物、探针与核酸模板的稳定高效结合,确保高效的PCR反应(参见"Unique PCR buffer")此外,HotStarTaq Plus DNA Polymerase要求严谨的热启动,可阻止非特异性产物的形成。
| 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 Multiplex 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 |
| 合成的MP因子 |
在单管中可靠的对至多4个基因进行多重分析 |
| 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 |
操作流程
QuantiFast Multiplex PCR Kit含有即用型预混液,无需优化反应及循环条件。只需将cDNA和引物探针对加入预混液中,按照操作手册进行实验,即可快速获得可靠的结果,适用于所有real-time PCR仪。试剂盒提供两种形式:预混液中含有或不含荧光校正用的ROX染料,可用于所有PCR仪(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。
| 混合在预混液中 |
QuantiFast Multiplex PCR Kit |
除Applied Biosystems 7500外的所有Applied Biosystems仪器 |
| 单独装在管中 |
QuantiFast Multiplex PCR +R Kit |
Applied Biosystems 7500、Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche、Agilent和其他供应商的PCR仪 | 为获得最佳的两步法real-time RT-PCR结果,我们建议使用QuantiTect Reverse Transcription Kit合成cDNA。该试剂盒可在20分钟进行快速cDNA合成,并去除基因组DNA污染。 QuantiFast Probe Assays利用序列特异性水解探针检测技术,是适合全基因组的预制试剂。 随QuantiFast Multiplex PCR Kit提供,可确保在双染两步法real-time RT-PCR反应中获得可靠结果。
应用
QuantiFast Multiplex PCR Kit可用在各种real-time PCR仪上对cDNA进行多重基因表达分析,适用仪器包括Agilent、Applied Biosystems、Bio-Rad、Cepheid、Eppendorf和Roche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 Multiplex PCR Kit。
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For quantitative, multiplex, real-time PCR and two-step RT-PCR with fast cycling using sequence-specific probes
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Significantly reduced PCR times.
QuantiFast Multiplex Kits reduce total PCR run time by up to 50% in real-time two-step RT-PCR (40 cycles run; comparison with QuantiTect Multiplex Kits). I: iCycler iQ; L1: LightCycler 480; L2: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500 Fast System; M: Mx3005P.
Sensitive duplex PCR and wide dynamic range.
Duplicate reactions were run on the Applied Biosystems 7500 Fast System using a DNA template mix providing 108 copies of β-actin (data shown in insets) and 106 to 10 copies of RPS27A (a ribosomal protein). The [A] QuantiFast Multiplex PCR +R Kit showed clearly higher sensitivity than the [B] duplex PCR kit from Supplier AII, enabling the cycler in fast-cycling mode to detect 10 copies of target and quantify over 6 log dilutions of template.
Comparable results in triplex and singleplex PCR.
Triplex and singleplex PCR were carried out on the LightCycler 480 using the QuantiFast Multiplex PCR +R Kit and Primer Express designed TaqMan assays for [A] RPS27A (a ribosomal protein), [B] GAPDH (a housekeeping gene), and [C] UBC (a housekeeping gene). The template was HeLa cell cDNA (10 ng, 1 ng, or 0.1 ng), and reactions were run in duplicate. The comparable CT values for triplex PCR (colored curves) and singleplex PCR (gray curves) demonstrate the reliability of triplex PCR with the QuantiFast Multiplex PCR +R Kit when analyzing targets of differing abundance.
Uncompromised sensitivity.
4-plex, real-time PCR was carried out using the QuantiFast Multiplex PCR +R Kit and Primer Express designed TaqMan assays on the Applied Biosystems 7500 Fast System. Triplicate reactions were run using 10 ng Ramos cell line cDNA.
Fast primer annealing.
[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the 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 annealing time.
QIAGEN multiplex kits.
QuantiFast 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).
Unique PCR buffer.
[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.
Linear CT values over twofold decreases in template.
Duplex and singleplex PCR were carried out using the QuantiFast Multiplex PCR +R Kit and assays for the t(8;14) chromosomal translocation and for GAPDH on the Applied Biosystems 7500 Fast System. Quadruplicate reactions were run using genomic DNA from the Ramos cell line as template (twofold dilutions from 10 ng to 0.625 ng). CT values increased linearly by 1 CT value with decrease in template dilution for both the [A] singleplex and [B] duplex reactions, demonstrating the ability of the kit to precisely discriminate between small differences in template amount.
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