使用序列特异性探针进行一步法real-time RT-PCR,用于基因表达分析

  • 灵敏检测低拷贝数模板
  • 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
  • 无需优化反应和循环条件

QuantiTect Probe RT-PCR Kit使用序列特异性探针通过一步法real-time PCR对RNA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RT-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。

Product Product no. Cat. no. List price:
 
 
Show details
    varies
Can't order online?
To place an order via phone, email or for requesting a quote, please provide the product’s name, number and catalog number.
Product Cat. no. List price:
QuantiTect Probe RT-PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe RT-PCR Master Mix, 100 µl QuantiTect RT Mix, 2 x 2 ml RNase-Free Water
204443
Inquire
QuantiTect Probe RT-PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Probe RT-PCR Master Mix, 0.5 ml QuantiTect RT Mix, 20 ml RNase-Free Water
204445
Inquire
The QuantiTect Probe RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
  • Main Image Navi
One-step RT-PCR.|Highly specific amplification.|High sensitivity and efficiency, and a wide dynamic range.|One-step RT-PCR with comparable performance to two-step RT-PCR - A.|One-step RT-PCR with comparable performance to two-step RT-PCR - B.|Specific primer annealing.|
The QuantiTect Probe RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and RNA template to the ready-to-use RT-PCR master mix, and start the reaction on any real-time cycler (see also table "Components of the QuantiTect Probe RT-PCR Kit").|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.|Tenfold serial dilutions of HeLa cell RNA (100 ng to 1 pg) were analyzed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe RT-PCR Kit with primers and a HEX labeled probe specific for human 28S rRNA. High PCR efficiency over 6 logs of template dilution was achieved.|Tenfold serial dilutions of cDNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for IL1R2 (interleukin 1 receptor, type II). Two-step RT-PCR was performed using the QuantiTect Probe PCR Kit (PCR efficiency: 101%).|Tenfold serial dilutions of RNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for ILR2 (interleukin 1 receptor, type II). One-step RT-PCR was performed using the QuantiTect Probe RT-PCR Kit (PCR efficiency: 104%).|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 RT-PCR Kit含有独特的RT-PCR缓冲液,可促使引物高度特异性的退火并与PCR模板结合。含有的HotStarTaq DNA Polymerase与其他聚合酶相比,具有严格的热启动(参见"Highly specific amplification")。

QuantiTect Probe RT-PCR Kit包括HotStarTaq DNA聚合酶和独特的RT-PCR缓冲液,能够高灵敏度的定量检测低拷贝的RNA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"High sensitivity and efficiency, and wide dynamic range")。在同一个管中连续进行逆转录和PCR不会影响灵敏度,通过与两步法real-time RT-PCR的CT值对比可证明(参见"One-step RT-PCR with comparable performance to two-step RT-PCR — A"和"One-step RT-PCR with comparable performance to two-step RT-PCR — B"及下表)。

一步法RT-PCR和两步法RT-PCR有相当的表现
cDNA/RNA的量 平均CT 平均CT
(两步法RT-PCR) (一步法RT-PCR)
100 ng 24.66 24.66
10 ng 28.15 27.84
1 ng 31.42 31.49
0.1 ng 34.80 34.56
0.01 ng 37.78 37.66
CT值来自图"One-step RT-PCR with comparable performance to two-step RT-PCR — A" 和"One-step RT-PCR with comparable performance to two-step RT-PCR — B"的两个扩增曲线。
Principle

QuantiTect Probe RT-PCR Kit含有优化的即用型预混液,可使用序列特异性探针对RNA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 RT-PCR Kit独特的PCR缓冲液含有浓度平衡的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,优化的逆转录酶混合液可从不同量的RNA模板合成cDNA,而HotStarTaq DNA Polymerase具有严格的热启动,可防止非特异性产物的形成。

QuantiTect Probe RT-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 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 Probe 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
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 Probe RT-PCR Kit无需优化反应条件。只需将引物、探针和RNA模板加入到即用型RT-PCR预混液中,即可开始反应(参见"One-step RT-PCR")。按照使用手册中的实验方案操作,即可快速获得可靠的结果,适用于各种real-time PCR仪。如果需要,可使用UNG进行预处理,去除上一反应残留的PCR产物。

Applications

QuantiTect Probe 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,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 RT-PCR Kit。

Feature
Specifications
Applications Real-time quantification of RNA targets
Reaction type One-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA targets
Single or multiplex Single
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX

You are not authorized to download the resource

FAQs
27
FAQ ID -1056
View
FAQ ID -1066
View
FAQ ID -1085
View
FAQ ID -1086
View
FAQ ID -1438
View
FAQ ID -1441
View
FAQ ID -1446
View
FAQ ID -1447
View
FAQ ID -1451
View
FAQ ID -283
View
FAQ ID -2117
View
FAQ ID -2119
View
FAQ ID -2134
View
FAQ ID -2135
View
FAQ ID -2371
View
FAQ ID -2655
View
FAQ ID -2682
View
FAQ ID -539
View
FAQ ID -540
View
FAQ ID -542
View
FAQ ID -564
View
FAQ ID -584
View
FAQ ID -587
View
FAQ ID -589
View
FAQ ID -729
View
FAQ ID-751
View
FAQ ID -784
View
Kit Handbooks
1
For quantitative, real-time one-step RT-PCR using sequence-specific probes
Show details