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.
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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
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204443
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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
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204445
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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。
See trademarks.
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.|
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.
| 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.
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特点
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参数
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应用
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Real-time quantification of RNA targets
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反应类型
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One-step RT-PCR
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real-time或终点法PCR
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Real-time
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样本/目标类型
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RNA targets
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单一或多重
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Single
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SYBR Green I或序列特异性探针
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Sequence-specific probes
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热循环仪
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All real-time cyclers (e.g. LC, RG, ABI)
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有/无ROX
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With ROX
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For quantitative, real-time one-step RT-PCR using sequence-specific probes
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图片
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。
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。
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。
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%)。
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%)。
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。
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