QuantiTect Multiplex RT-PCR Kits

使用序列特异性探针进行多重一步法定量RT-PCR，用于基因表达分析

无需优化的多重分析
单次反应分析多个目标序列
灵敏地检测低至10个拷贝的目标序列
可靠的定量检测目标序列和对照基因
在单管中检测对照基因和至多3个目标序列

QuantiTect Multiplex RT-PCR Kit利用多重一步法real-time RT-PCR技术，可在单管中对至多5个目标RNA进行可靠的定量检测。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qRT-PCR，无需优化。dNTP预混液中含有dUTP，因此可选择性进行UNG处理。有两种规格的试剂盒：QuantiFast Multiplex RT-PCR Kit适用于需要ROX染料进行荧光信号校准的PCR仪，QuantiFast Multiplex RT-PCR NoROX 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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Product	Cat. no.	List price:
QuantiTect Multiplex RT-PCR Kit (200) For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Multiplex RT-PCR Master Mix (with ROX dye), 100 µl QuantiTect Multiplex RT Mix, 2 x 2 ml RNase-Free Water	204643	£632.00	Add to cart
QuantiTect Multiplex RT-PCR Kit (1000) For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex RT-PCR Master Mix (with ROX dye), 0.5 ml QuantiTect Multiplex RT Mix, 20 ml RNase-Free Water	204645	£2,530.00	Add to cart
QuantiTect Multiplex RT-PCR NR Kit (200) For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Multiplex RT-PCR NoROX Master Mix (without ROX dye), 100 µl QuantiTect Multiplex RT Mix, 2 x 2 ml RNase-Free Water	204843	£632.00	Add to cart
QuantiTect Multiplex RT-PCR NR Kit (1000) For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex RT-PCR NoROX Master Mix (without ROX dye), 0.5 ml QuantiTect Multiplex RT Mix, 20 ml RNase-Free Water	204845	£2,505.00	Add to cart

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QuantiTect Multiplex 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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Comparable amplification in triplex PCR and singleplex PCRs.|Detection of down to 10 copies of target RNA in duplex PCR.|Comparable amplification in 4-plex PCR and singleplex PCRs.|QIAGEN multiplex kits.|Unique PCR buffer.|

Triplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The template was 20 ng total RNA from the Burkitt's lymphoma cell line Ramos. Reactions were performed in triplicate. 28S rRNA was detected using a HEX labeled probe. POLD3 (accessory subunit of DNA polymerase delta 3) was detected using a FAM labeled probe. CDK2 (cell cycle-dependent kinase 2) was detected using a Cy5 labeled probe. For comparison, the targets were also quantified by singleplex, real-time one-step RT-PCR (black curves). Curves for triplex PCR and singleplex PCRs overlap, demonstrating comparable amplification (i.e., equivalent C_T values).|Duplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The target was [A] 1000, 100, or 10 copies of an in vitro transcript of a viral gene, each spiked with [B] 1000 copies of synthetic internal control RNA. Reactions were performed in replicate. Target RNA was detected using a FAM labeled probe. Internal control was detected using a HEX labeled probe. 7 replicates of 10 copies of target RNA were reproducibly detected.|4-plex, real-time one-step RT-PCR was performed on the LightCycler 2.0 using the QuantiTect Multiplex RT-PCR NR Kit and TaqMan probes. The template was 10, 1, or 0.1 ng of total RNA purified from K562 cells. [A] c-myc (a proto-oncogene) was detected using a FAM labeled probe. [B] GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was detected using a HEX labeled probe. [C] HSP89 (a heat shock protein) was detected using a Texas Red labeled probe. [D] H28S (28S rRNA) was detected using an Alexa Fluor 660 labeled probe. For comparison, the targets were also quantified by singleplex, real-time one-step RT-PCR (black curves). Curves for 4-plex and singleplex PCRs overlap, showing comparable amplification (i.e., equivalent C_T values).|QuantiTect Multiplex RT-PCR Kits provide a simple procedure for quantitative, multiplex, real-time one-step RT-PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, probes, 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 DNA Polymerase.|

Performance

QuantiTect Multiplex RT-PCR Kit可灵敏地检测低至10个拷贝的目标序列（参见"Detection of down to 10 copies of target RNA in duplex PCR"）。即使用4重检测法定量起始拷贝数相差达100,000倍的转录本也可获得理想的实验结果（参见"Comparable amplification in 4-plex PCR and singleplex PCRs"）。

通过在同一孔板或试管内同时定量分析靶基因和内源对照基因，可精确定量检测基因表达。使用QuantiTect Multiplex RT-PCR Kit时，靶基因在多重PCR中的C_T值与其在单一PCR对照实验中C_T值一致（参见"Comparable amplification in triplex PCR and singleplex PCRs"）。这说明不同的靶基因在同一个多重PCR反应体系中可被高效、高灵敏度地扩增而不相互干扰。

Principle

通过在同一孔板或试管内同时定量分析靶基因和内源对照基因，可精确定量检测基因表达。QuantiTect Multiplex RT-PCR Kit确保在多重一步法RT-PCR中一次成功（参见"QIAGEN multiplex kits"）。优化的预混液确保PCR产物在多重反应中的扩增效率和灵敏度与在单一反应中相同。此试剂盒可检测低至10个拷贝的靶基因。

在同一反应液中扩增内参和靶基因，可减少手工误差，提高基因定量分析的可靠性。QuantiTect Multiplex RT-PCR Master Mix含有浓度平衡的K⁺、NH₄⁺和独特合成的MP因子，可促使引物和探针与核酸模板稳定、高效的退火，提高PCR效率（参见"Unique PCR buffer"）。此外，优化的反转录酶混合液可从不同量的RNA模板合成cDNA，而HotStarTaq Plus DNA Polymerase具有严谨的热启动，防止非特异性产物的产生。

2x QuantiTect Multiplex RT-PCR Kit的组份
组份	特点	优势
HotStarTaq DNA Polymerase	95ºC孵育15分钟即可活化	室温下即可构建qPCR反应体系
QuantiTect Multiplex RT-PCR Buffer	NH₄⁺和K⁺的平衡组合	特异性引物退火确保PCR结果可靠
QuantiTect Multiplex RT-PCR Buffer	合成的Factor MP	在单管内对4个基因进行可靠的多重分析
dNTP mix	含有dUTP，会部分替代dTTP，可进行UNG处理	可选择进行UNG处理，以消除PCR产物中的残余污染物
ROX dye*	在Applied Biosystems或Agilent的PCR仪上进行荧光信号校准	可在需要ROX染料的PCR仪上进行准确的定量检测。不会干扰其他real-time PCR仪
Omniscript and Sensiscript Reverse Transcriptases	多种酶，对RNA亲和性高	RNA、甚至是具有复杂二级结构的RNA均可实现转录

Procedure

QuantiTect Multiplex RT-PCR Kit含有即用型预混液，无需对反应液和循环条件进行优化。操作手册提供了适用于各种real-time PCR仪的实验方案，并列有推荐的染料。如果需要，反应液可用尿嘧啶DNA糖基化酶（UNG）（不提供）预处理，以去除PCR产物中残留的污染物。

可选预混液有或没有ROX染料的试剂盒，确保能在各种real-time PCR仪上使用（见表）。ROX浓度已优化，可通过自动数据分析对低拷贝数进行检测。

选择合适的QuantiTect Multiplex RT-PCR Kit
ROX染料	试剂盒	兼容的PCR仪
预混液中含有ROX染料	QuantiTect Multiplex RT-PCR Kit	Applied Biosystems PCR仪
预混液中不含ROX染料	QuantiTect Multiplex RT-PCR NR Kit	Rotor-Gene系列PCR仪器和Bio-Rad、Cepheid、Eppendorf、Roche、Agilent等PCR仪

Applications

QuantiTect Multiplex RT-PCR Kit可用在各种real-time PCR仪上对RNA目标序列进行基因表达分析。仪器包括Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、Roche和Agilent的仪器。在Rotor-Gene Q实时荧光定量PCR分析仪上使用时，我们推荐专门为快速PCR研发的Rotor-Gene Multiplex RT-PCR Kit。

Feature	Specifications
Applications	Real-time quantification of RNA targets in a multiplex format
Description	For quantitative, multiplex, real-time one-step RT-PCR using sequence-specific probes
Reaction type	Real-time one-step RT-PCR
Real-time or endpoint	Real-time
Sample/target type	RNA targets
Single or multiplex	Multiplex
SYBR Green I or sequence-specific probes	Sequence-specific probes
Thermal cycler	Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
With or without ROX	With ROX or without

Brochures & Guides

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	(EN) - Analyzing Gene Expression and Regulation	Show details
	Critical Factors for Successful Real-Time PCR - (EN)	Show details

FAQs

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	What should I use as a standard for absolute quantification in real-time PCR? FAQ ID -1085	View
	How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits? FAQ ID -1438	View
	How do QuantiFast PCR Kits compare to QuantiTect PCR Kits for quantitative real-time PCR? FAQ ID -1441	View
	What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits? FAQ ID -2119	View
	Can Uracil-N-Glycosylase (UNG) be purchased separately, independent of QuantiTect PCR Kits? FAQ ID -2134	View
	Do you have any information or guidelines regarding the choice of reference genes for real-time PCR? FAQ ID -2371	View
	How important is the RNA purification process, for obtaining reliable qRT-PCR results? FAQ ID -2655	View
	What is the threshold cycle or Ct value? FAQ ID -2682	View
	Why does my realtime PCR assay quality decrease over time? FAQ ID -589	View
	Why should DNA or cDNA targets be less than 250 bp long for real-time PCR? FAQ ID-751	View
	How should fluorescent labeled probes be stored? FAQ ID -784	View

Technical Information

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	Overcoming the challenges of real-time, multiplex PCR - (EN)	Show details

Quick-Start Protocols

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	QuantiTect Multiplex RT-PCR Kit (EN)	Show details
	QuantiTect Multiplex RT-PCR NR Kit (EN)	Show details

Kit Handbooks

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	(EN) - QuantiTect Multiplex RT-PCR Handbook QuantiTect Multiplex RT-PCR Kit - with master mix containing ROX passive reference dye QuantiTect Multiplex RT-PCR NR Kit - with master mix free of ROX passive reference dye For gene expression analysis by quantitative, multiplex, real-time one-step RT-PCR using sequence-specific probes	Show details

Safety Data Sheets

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	Safety Data Sheets Download Safety Data Sheets for QIAGEN product components.	View
	Safety Data Sheets Download Safety Data Sheets for QIAGEN product components.	View

Safety Data Sheets

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	MSDS QuantiTect Multiplex RT-PCR Kit (200)
	MSDS QuantiTect Multiplex RT-PCR Kit (1000)
	MSDS QuantiTect Multiplex RT-PCR NR Kit (200)
	MSDS QuantiTect Multiplex RT-PCR NR Kit (1000)
	CofA

Images

Comparable amplification in triplex PCR and sigleplex PCRs on the Applied Biosystems 7500.

Comparable amplification in triplex PCR and singleplex PCRs.

Detection of down to 10 copies of target RNA in duplex PCR.

Duplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The target was [A] 1000, 100, or 10 copies of an in vitro transcript of a viral gene, each spiked with [B] 1000 copies of synthetic internal control RNA. Reactions were performed in replicate. Target RNA was detected using a FAM labeled probe. Internal control was detected using a HEX labeled probe. 7 replicates of 10 copies of target RNA were reproducibly detected.

Comparable amplification in 4-plex PCR and singleplex PCRs.

4-plex, real-time one-step RT-PCR was performed on the LightCycler 2.0 using the QuantiTect Multiplex RT-PCR NR Kit and TaqMan probes. The template was 10, 1, or 0.1 ng of total RNA purified from K562 cells. [A] c-myc (a proto-oncogene) was detected using a FAM labeled probe. [B] GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was detected using a HEX labeled probe. [C] HSP89 (a heat shock protein) was detected using a Texas Red labeled probe. [D] H28S (28S rRNA) was detected using an Alexa Fluor 660 labeled probe. For comparison, the targets were also quantified by singleplex, real-time one-step RT-PCR (black curves). Curves for 4-plex and singleplex PCRs overlap, showing comparable amplification (i.e., equivalent C_T values).

Successful Multiplex PCR-Based Genotypic Analysis without the Need for Optimization

QIAGEN multiplex kits.

QuantiTect Multiplex RT-PCR Kits provide a simple procedure for quantitative, multiplex, real-time one-step RT-PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, probes, 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 Type-it Microsatellite PCR Buffer Promotes Stable and Efficient Annealing

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 DNA Polymerase.