使用序列特异性探针进行两步法实时定量RT-PCR,用于基因表达分析

  • 高灵敏度检测低拷贝的模板
  • 准确检测各种起始量的模板
  • 可用各种序列特异性探针,用于各种PCR仪
  • 提供含有或不含尿嘧啶DNA糖基化酶(UNG)两种包装
  • 无需优化反应和循环条件

QuantiTect Probe PCR Kit使用序列特异性探针通过两步法real-time PCR对cDNA目标序列进行灵敏的定量分析。即用型预混液使用热启动和独特的PCR缓冲液,无需优化即可在各种real-time PCR仪上进行高度灵敏的定量PCR。dNTP混合液含有dUTP,可选择性进行UNG处理。为便于使用,预混液可储存在2–8°C。

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QuantiTect Probe PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 2 x 2 ml RNase-Free Water
204343
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QuantiTect Probe PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Probe PCR Master Mix, 20 ml RNase-Free Water
204345
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QuantiTect Probe PCR +UNG Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 100 ul UNG Solution, 2 x 2 ml RNase-Free Water
204363
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QuantiTect 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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Effective UNG digestion.|Two-step RT-PCR.|Highly specific amplification.|Wide dynamic range in two-step PCR.|Wide dynamic range in real-time PCR.|High sensitivity and efficiency, and wide dynamic range.|Specific primer annealing.|
106 copies of two dUMP-containing PCR amplicons were treated with or without UNG and then amplified by real-time PCR. UNG was from various suppliers, and real-time PCR was performed using the master mix from the QuantiTect Probe PCR +UNG Kit. ΔCT on the Y-axis indicates CT values for non-UNG-treated samples subtracted from CT values for UNG-treated samples. The UNG supplied with the QuantiTect Kit provided the greatest ΔCT (9-12 cycles). This indicates that QIAGEN UNG digests carryover PCR products more effectively than UNG from other suppliers.|The QuantiTect Probe PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and cDNA template to the ready-to-use PCR master mix, and start the reaction on any real-time cycler (see also the table "Components of 2x QuantiTect Probe PCR Master Mix").|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Duplicate reactions were performed on the Mx3005P using tenfold dilutions of human keukocyte cDNA (10 ng to 0.01 ng) and a TaqMan assay for IL1R2 (a cytokine). The QuantiTect Probe PCR Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 101%.|Probe-based real-time PCR with UNG pretreatment was carried out using the QuantiTect Probe PCR +UNG Kit. Reactions were run in duplicate on the ABI PRISM 7900 using 10-fold dilutions of human leukocyte cDNA (100 ng to 10 pg) and a TaqMan assay for IL8 (a cytokine). The amplification plots were evenly spaced, leading to a high PCR efficiency of 94%.|Tenfold serial dilutions of leukocyte cDNA (100 ng to 1 pg) were analyzed in duplicate on the Mx3005 using the QuantiTect Probe PCR Kit with primers and a FAM-labeled probe specific for IL8 (interleukin 8). A PCR efficiency of 97% over 6 logs of template dilution was achieved, and as little as 1 pg of IL8 transcript was sensitively detected.| 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. |
Performance

QuantiTect Probe PCR Kit配合QuantiTect Reverse Transcription Kit可得到灵敏、可靠的结果(参见"High sensitivity and efficiency, and wide dynamic range")。独特的PCR缓冲液组分使QuantiTect Probe PCR Kit能够高灵敏度的定量检测低拷贝的DNA靶序列,在宽泛的范围内符合线性规律,实现准确定量检测(参见"Wide dynamic range in two-step RT-PCR")。与其他聚合酶相比,HotStarTaq DNA Polymerase具有严谨的热启动,进一步提高了PCR反应的特异性(参见"Highly specific amplification")。

PCR污染的主要来源是PCR产物中的残余DNA污染。在PCR前使用UNG预处理,可确保污染的PCR产物不会影响后续的PCR。QuantiTect Probe PCR +UNG Kit中经特别优化的UNG溶液配合经验证的PCR预混液可有效避免PCR产物污染,并对靶序列进行可靠的定量(参见"Effective UNG digestion"和"Wide dynamic range in real-time PCR")。

Principle

QuantiTect Probe PCR Kit含有优化的即用型预混液,可使用序列特异性探针对cDNA靶序列进行高特异性和灵敏度的real-time定量分析。此试剂盒适用于各种类型的序列特异性探针,包括水解探针检测(如TaqMan和其他双标记探针)、FRET探针和Molecular Beacons。QuantiTect Probe PCR Kit独特的PCR缓冲液含有K+和NH4+,能促使特异性引物退火,获得高PCR特异性和灵敏度(参见"Specific primer annealing")。此外,HotStarTaq DNA Polymerase具有严格的热启动,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,保证污染的PCR产物不会影响后续的PCR反应。

2x QuantiTect Probe 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 Probe PCR Buffer NH4+和K+的平衡组合 特异性引物退火确保PCR结果可靠
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
Procedure

QuantiTect Probe PCR Kit无需进行繁琐耗时的反应条件优化。只需简单地将引物、探针和cDNA模板加入到即用型PCR预混液中,即可开始反应(参见"Two-step RT-PCR")。使用操作手册中的实验方案可在任何real-time PCR仪上得到快速、可靠的结果。如果需要,反应液可用尿嘧啶DNA糖基化酶(UNG)预处理,以去除PCR产物中残留的污染。

为在两步法real-time RT-PCR中获得理想结果,我们推荐使用QuantiTect Reverse Transcription Kit合成cDNA。此试剂盒可在20分钟内快速合成cDNA,并可去除基因组DNA污染。

Applications

QuantiTect Probe PCR Kit可用于在各种real-time PCR仪上对cDNA目标序列进行基因表达分析,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,我们推荐专门为快速PCR研发的Rotor-Gene Probe PCR Kit。

Feature
Specifications
Applications Real-time quantification of DNA, cDNA, or RNA targets
Reaction type PCR and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type DNA, cDNA, RNA
Single or multiplex Single
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

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Kit Handbooks
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For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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For quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
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