QuantiTect Probe RT-PCR Kit

For one-step qRT-PCR using sequence-specific probes for gene expression analysis

  • Highly sensitive detection of low-copy targets
  • Accurate quantification over several logs of template
  • Use of any sequence-specific probe on any real-time cycler
  • No need to optimize reaction and cycling conditions

QuantiTect Probe RT-PCR Kits enable sensitive quantification of RNA targets by real-time one-step PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.

产品 货号 目录价:
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
询价
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
询价

QuantiTect Probe RT-PCR Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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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

The QuantiTect Probe RT-PCR Kit has a unique RT-PCR buffer that promotes highly specific annealing of primers and probes to the PCR template. It contains HotStarTaq DNA Polymerase, which provides the most stringent hot start compared with other polymerases (see figure "Highly specific amplification").

HotStarTaq DNA Polymerase and the unique composition of the RT-PCR buffer enable the QuantiTect Probe RT-PCR Kit to provide sensitive quantification of low-copy RNA targets, as well as accurate quantification over a wide linear range (see figure "High sensitivity and efficiency, and wide dynamic range"). Performing reverse transcription and PCR sequentially in the same tube does not impair sensitivity, as demonstrated by CT values that are comparable to those achieved in real-time two-step RT-PCR (see figures "One-step RT-PCR with comparable performance to two-step RT-PCR — A" and "One-step RT-PCR with comparable performance to two-step RT-PCR — B", and table).

One-step RT-PCR with comparable performance to two-step RT-PCR.
Amount of cDNA/RNAMean CT value Mean CT value
(two-step RT-PCR) (one-step 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 values from the 2 amplification plots shown in figures "One-step RT-PCR with comparable performance to two-step RT-PCR — A" and "One-step RT-PCR with comparable performance to two-step RT-PCR — B".
Principle

QuantiTect Probe RT-PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using sequence-specific probes. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probes (e.g., TaqMan® and other dual-labeled probes), FRET probes, and Molecular Beacons. QuantiTect Probe RT-PCR Kits contain a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, an optimized mix of reverse transcriptases enables cDNA synthesis from a wide range of RNA template amounts, while HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

QuantiTect Probe RT-PCR Master Mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.

Components of 2x QuantiTect Probe RT-PCR Kit
Component Features Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect Probe RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions Eliminates contamination from carryover of PCR products by optional UNG treatment
ROX dye For normalization of fluorescent signals on Applied Biosystems and, optionally,  Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Omniscript and Sensiscript Reverse Transcriptases Special blend of enzymes with high affinity for RNA RNA can be transcribed, even through complex secondary structures
Procedure

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 (see flowchart "One-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions. 

Applications

QuantiTect Probe RT-PCR Kits can be used for gene expression analysis of RNA targets on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Probe RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

特点
参数
应用 Real-time quantification of RNA targets
反应类型 One-step RT-PCR
real-time或终点法PCR Real-time
样本/目标类型 RNA targets
单一或多重 Single
SYBR Green I或序列特异性探针 Sequence-specific probes
热循环仪 All real-time cyclers (e.g. LC, RG, ABI)
有/无ROX With ROX

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试剂盒操作手册
1
For quantitative, real-time one-step RT-PCR using sequence-specific probes
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安全数据表
1
Download Safety Data Sheets for QIAGEN product components.
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图片
PCR and two-step RT-PCR.
一步法RT-PCR。
QuantiTect Probe RT-PCR Kit避免了繁琐且耗时的反应条件优化过程。只需将引物、探针和cDNA模板加入即用型RT-PCR预混液,在任意一台real-time循环仪上开始反应即可。
Highly specific amplification.
高度特异性扩增。
相比其他DNA聚合酶,只有HotStarTaq DNA Polymerase结合独特的缓冲液可以特异性扩增497 bp的片段(在1 µg人基因组DNA背景下的50个拷贝的HIV-pol基因重组体)。M:分子量标准。
Wide dynamic range in one-step RT-PCR
高度灵敏且高效的反应。
在ABI PRISM 7900上使用QuantiTect Probe PCR Kit及人28S rRNA特异性的引物和HEX标记的探针分析10倍梯度稀释的HeLa细胞RNA(100 ng至1 pg)。在6 log模板稀释度范围内具有高PCR效率。
One-Step RT-PCR with Comparable Performance to Two-Step RT-PCR
一步法RT-PCR的表现可与两步法RT-PCR相媲美— A。
在Mx3005上使用IL1R2(白介素1受体II型)特异性的引物和FAM标记的探针分析10倍梯度稀释的白细胞cDNA(100 ng至1 pg),反应重复两次。使用QuantiTect Probe PCR Kit进行两步法RT-PCR(PCR效率:101%)。
One-step RT-PCR with comparable performance to a two-step RT-PCR
一步法RT-PCR的表现可与两步法RT-PCR相媲美— B。
在Mx3005上使用ILR2(白介素1受体II型)特异性的引物和FAM标记的探针分析10倍梯度稀释的白细胞RNA(100 ng至1 pg),反应重复两次。使用QuantiTect Probe RT-PCR Kit进行一步法RT-PCR(PCR效率:104%)。
Specific primer annealing using a balanced combination of K+ and NH4+ ions
退火时引物特异性结合。
退火时,缓冲液中浓度平衡的KCl和(NH4)2SO4促进引物和探针特异性与模板结合。K+结合到双链DNA上的磷酸基团,稳定引物和探针。NH4+则破坏弱的错配碱基之间的氢键。