使用序列特异性探针进行两步法多重定量RT-PCR,用于基因表达分析

  • 无需优化的多重PCR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

QuantiTect Multiplex PCR Kit可通过多重两步法real-time PCR在单管中对5个cDNA进行可靠的定量检测。即用型预混液具有热启动和独特的PCR缓冲液体系,无需优化即可在各种real-time PCR仪上进行高度灵敏、可靠的多重定量PCR。dNTP混合液含有dUTP,可选择性进行UNG处理。该试剂盒有两种规格:QuantiTect Multiplex PCR Kit适用于需要ROX染料进行荧光校正的PCR仪,QuantiTect Multiplex PCR NoROX Kit适用于其他PCR仪。为便于使用,预混液可储存在2–8°C。

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QuantiTect Multiplex PCR Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect Multiplex PCR Master Mix (with ROX dye), 2 ml RNase-Free Water
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QuantiTect Multiplex PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Multiplex PCR Master Mix (with ROX dye), 2 x 2 ml RNase-Free Water
204543
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QuantiTect Multiplex PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex PCR Master Mix (with ROX dye), 20 ml RNase-Free Water
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QuantiTect Multiplex PCR NoROX Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye), 2 ml RNase-Free Water
204741
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QuantiTect Multiplex PCR NoROX Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye), 2 x 2 ml RNase-Free Water
204743
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QuantiTect Multiplex PCR NoROX Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye), 20 ml RNase-Free Water
204745
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QuantiTect Multiplex 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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Equivalent CT values in triplex and singleplex PCR.|Comparable results between 4-plex PCR and corresponding singleplex PCRs.|Successful quantitative triplex qPCR of low and high abundance targets.|Detection of 10 copies of target gene with excess reference gene.|QIAGEN multiplex kits.|Unique PCR buffer.|
Dilutions of cDNA template (100 ng, 11.11 ng, 1.23 ng, and 0.14 ng) were analyzed by triplex PCR (colored curves) and by singleplex PCR (gray curves) on the iCycler iQ. TaqMan probes labeled with FAM, HEX, or Cyanine 670 dye were used. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA.)|GAPDH, 18S, HSP, and H28S sequences were amplified either together in 4-plex PCR or in singleplex PCRs on the Rotor-Gene 3000 system using the QuantiTect Multiplex PCR NoROX Kit. The templates were 100, 10, 1, and 0.1 ng of human leukocyte cDNA. Reactions were performed in triplicate. TaqMan probes labeled with FAM, HEX, Texas Red, or Cyanine 670 dye were used. 4-plex PCR and singleplex PCR data are displayed on the amplification plots, with the curves for the singleplex PCRs colored in gray. The plots show reliable multiplex quantification over a wide range of template amounts as well as good reproducibility within each set of triplicates. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S rRNA; HSP: a heat shock protein; H28S: 28S rRNA.|Three sequences (CSBG, GAPDH, and HSP) were amplified either together in triplex PCR or in singleplex PCRs on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit. Three template mixes with varying amounts of human genomic DNA (for CSBG amplification), linearized plasmid (for GAPDH amplification), and human leukocyte cDNA (for HSP amplification) were prepared. Reactions were performed in duplicate. TaqMan probes labeled with FAM, HEX, or Texas Red dye were used. The amplification plot shows detection of CSBG in the triplex PCRs (Blue: 1400 copies; Green: 140 copies; Red: 14 copies).|[A] Duplex PCR was performed on the ABI PRISM 7700 using either the QuantiTect Multiplex PCR Kit or a kit from Supplier AII. Variable amounts of t(8;14) translocation sequence (50,000, 5000, 500, 50, or 10 copies) were coamplified with a constant amount of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) sequence (107 copies). The t(8;14) translocation sequence was amplified from Ramos cell line genomic DNA using a FAM labeled TaqMan probe. The GAPDH sequence was amplified from a plasmid containing GAPDH cDNA sequence using a HEX labeled TaqMan probe. In contrast to the kit from Supplier AII, the QuantiTect Multiplex PCR Kit reliably quantified high to low amounts of t(8;14) translocation sequence (down to 10 copies, indicated with an arrow) in the presence of a high amount (107 copies) of GAPDH sequence. [B] The procedure described in [A] was repeated, with the exception that the target sequences were amplified separately in singleplex PCRs.|QuantiTect 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).|[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, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase.|
Performance

QuantiTect Multiplex PCR Master Mix确保在多重扩增反应液中PCR产物扩增的效率和灵敏度与单一扩增反应中相同(参见“三重和单一PCR反应的CΤ值比较”、"Comparable results with 4-plex and singleplex PCRs"和"Equivalent CT values in triplex PCR and singleplex PCR")。

三重和单一PCR反应的CΤ值比较
检测t(8;14) 易位序列(20拷贝) 检测GAPDH cDNA序列(106拷贝) 检测NFKB cDNA序列(拷贝数参见第一栏)
105拷贝NFKB的三重PCR 34.31 20.37 21.92
相应的单一PCR 34.07 20.54 21.83
拷贝NFKB的三重PCR 34.61 20.62 25.03
相应的单一PCR 34.00 20.46 25.19
103拷贝NFKB的三重PCR 35.17 19.94 28.38
相应的单一PCR 34.43 20.50 28.65
在ABI PRISM 7900上使用QuantiTect Multiplex PCR Kit对三个序列进行三重或单重PCR扩增。模板是来自Ramos细胞系的140 pg基因组DNA,并带有t(8;14)的易位(目标序列约20个拷贝);106拷贝数的含有人GADPH cDNA序列的线性质粒;105、104或103 拷贝数的含有人NFKB cDNA序列的质粒。通过一次实验模拟了对少量、大量和不同量的靶基因的定量检测。使用了标记有FAM、HEX或Bodipy TMR报告染料及BHQ淬灭序列的TaqMan探针。

此试剂盒可检测低至10个拷贝数的靶基因,即使同一反应液中对照基因的拷贝数是106倍以上(参见"Detection of 10 copies of target gene with excess reference gene"、"Successful quantitative triplex qPCR of low and high abundance targets"和“对高和低丰度靶基因的进行成功的三重real-time PCR检测”)。

对高和低丰度靶基因的进行成功的三重real-time PCR检测
检测CSBG 检测GAPDH 检测HSP
模板预混液1 1400拷贝 106拷贝 2 x 104拷贝
三重PCR的CT 27.73 18.69 23.59
单一PCR的CT 27.08 18.89 23.52
模板预混液2 140拷贝 106拷贝 2 x 103拷贝
三重PCR的CT 31.11 19.00 27.05
单一PCR的CT 30.66 18.61 26.97
模板预混液3 14拷贝 106拷贝 2 x 102拷贝
三重PCR的CT 34.74 18.98 30.71
单一PCR的CT 33.84 19.01 30.38
在LightCycler 2.0系统上使用QuantiTect Multiplex PCR NoROX Kit对三个序列(CSBG、GAPDH和HSP)进行了三重或多重PCR扩增。三种模板混有不同含量的人基因组DNA(用于CSBG扩增)、线性质粒(用于GADPH扩增)和人白细胞cDNA(用于HSP扩增)。反应含有两个平行。 TaqMan探针标记有FAM、HEX或Texas Red染料。表中显示了大约的目标序列拷贝数和三重及单重PCR的CT值。数据表明,在存在大量(106拷贝)GADPH序列的情况下检测少至14拷贝的CSBG序列,三重和单重PCR具有相当的CΤ值。CSBG:假丝酵母溶解β-葡聚糖;GADPH:甘油三磷酸脱氢酶;HSP:热激蛋白
Principle

使用QuantiTect Multiplex PCR Kit可确保两步法多重RT-PCR反应的成功(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对照和靶基因,可减少操作误差,提高基因定量分析的可靠性。QuantiTect Multiplex 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,HotStarTaq Plus DNA Polymerase具有严谨的热启动,可防止非特异性产物的形成。

QuantiTect Multiplex PCR Master Mix含有dUTP,可在PCR前使用尿嘧啶DNA糖基化酶(UNG)预处理,保证被污染的PCR产物不会影响后续的PCR反应。

QuantiTect Multiplex PCR Kit的组分
组分 特点 优势
HotStarTaq DNA Polymerase 在95ºC下15分钟即可活化 在室温下构建qPCR反应
QuantiTect Multiplex PCR Buffer 比例平衡的NH4+和K+ 特异性引物退火,确保可靠的PCR结果
Synthetic Factor MP 在同一管中对至多4个基因进行可靠的多重分析
dNTP混合液 包含dUTP,部分替代dTTP,可选用UNG处理 选用UNG处理,可消除PCR产物残留污染
ROX染料* 在Applied Biosystems或Agilent仪器上可标准化荧光信号 在需要ROX染料的循环仪上准确定量。不干扰在其他real-time循环仪上的反应。
* 如需查看预混液中是否很有ROX染料,请参见"Choosing the right QuantiTect Multiplex PCR Kit"。
Procedure

QuantiTect Multiplex 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,以去除之前反应残留的PCR产物。

可选预混液中含或不含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 PCR Kit
ROX染料 试剂盒兼容的循环仪
预混液中含有 QuantiTect Multiplex PCR Kit Applied Biosystems的循环仪
预混液中不含 QuantiTect Multiplex PCR NoROX Kit Rotor-Gene系列循环仪,Bio-Rad、Cepheid、Eppendorf、Roche、Agilent和其他供应商的循环仪

我们推荐使用QuantiTect Reverse Transcription Kit合成cDNA,可在两步法real-time RT-PCR中获得出色结果。该试剂盒可在20分钟内快速合成cDNA,并去除基因组DNA污染。

Applications

QuantiTect Multiplex PCR Kit可在各种real-time PCR仪上对cDNA进行基因表达分析,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,我们推荐使用专为快速PCR研发的Rotor-Gene Multiplex PCR Kit。

Feature
Specifications
Applications Quantification of cDNA or genomic DNA targets
Reaction type PCR product amplification
Real-time or endpoint Real-time
Sample/target type cDNA, DNA
Single or multiplex Multiplex
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler Most real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX or without

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Kit Handbooks
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QuantiTect Multiplex PCR Kit - with master mix containing ROX passive reference dye QuantiTect Multiplex PCR NoROX Kit - with master mix free of ROX passive reference dye For quantitative, multiplex, real-time PCR and two-step RT-PCR using sequence-specific probes  
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