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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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
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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
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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
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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
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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 CT 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 CT 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CT值与其在单一PCR对照实验中CT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
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
*如需查看预混液中是否很有ROX染料,请参见"Choosing the right QuantiTect Multiplex RT-PCR Kit"。
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

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Kit Handbooks
1
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
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