QIAGEN OneStep RT-PCR Kit
For highly sensitive and specific one-step RT-PCR
- Fast and easy one-tube setup
- Efficient one-step RT-PCR of any RNA template without optimization
- Unique enzyme mix for high specificity and sensitivity
- Balanced mixture of enzymes with optimized reverse-transcription buffer
The QIAGEN One-Step RT-PCR Kit provides a blend of Sensiscript and Omniscript Reverse Transcriptases, HotStarTaq DNA Polymerase, QIAGEN OneStep RT-PCR Buffer, a dNTP mix, and Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates. The easy one-tube setup and optimized components result in highly sensitive and successful results.
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QIAGEN OneStep RT-PCR Kit (25)
For 25 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 50 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 250 µl), dNTP Mix (1 x 50 µl, 10 mM each), 5x Q-Solution (1 x 400 µl), RNase-Free Water (1 x 1.9 ml)
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210210
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QIAGEN OneStep RT-PCR Kit (100)
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)
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210212
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QIAGEN OneStep 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。
OneStep RT-PCR procedure.|Highly specific RT-PCR using low amounts of template.|Efficient detection of viral RNA.|Influence of annealing temperature on specificity.|RT-PCR of GC-rich template.|Increased specific primer annealing.|
The QIAGEN OneStep RT-PCR Kit allows fast and easy RT-PCR setup. Whatever the application — virus detection, molecular diagnostics research, or gene expression analysis— just mix all components together in one tube and start the thermal-cycler program. The reaction mixture contains all of the reagents required for both reverse transcription and PCR; nothing needs to be added once the reaction has been started.|One-step RT-PCR was carried out using the indicated amounts of total RNA from HeLa cells and primers specific for α-catenin, amplifying a 690 bp product. All reactions were carried out following suppliers' instructions. M: 100 bp ladder.|A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplified from Sendai virus RNA isolated from persistently infected Vero cells. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit and the indicated number of viral genome copies. M: markers. (Data kindly provided by H. Rausch, Max Planck Institute for Biochemistry, Martinsried, Germany as part of the project "Experimental control of virological work at safety levels 2 and 3 in Bavaria," supported by the Bavarian Ministry of the Environment.)|One-step RT-PCR was performed using kits from the indicated suppliers over a range of annealing temperatures. A 1289 bp fragment from the human RCC1 gene was reverse transcribed and amplified from Hela RNA (arrow). M: markers. High levels of specific amplification without optimization were observed only with the QIAGEN OneStep RT-PCR Kit.|A 439 bp fragment of transcription factor TAFII100 mRNA (GC content: 75%) was reverse-transcribed and amplified from HeLa-cell total RNA. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit with (+) or without (–) Q-Solution. Q-Solution ensures specific amplification of GC-rich templates. M: markers.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specific primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific-to- nonspecific primer-template binding over a wide temperature range.|
Performance
The QIAGEN OneStep RT-PCR Kit provides a convenient format for highly sensitive and specific RT-PCR using any RNA template. The kit includes optimized components that allow both reverse transcription and PCR amplification to take place in the same reaction mix in a "one-step" reaction. A unique enzyme combination and specially developed reaction buffer ensure efficient, highly specific reverse transcription and PCR in one tube, without the need for optimization (see figures " Highly specific RT-PCR using low amounts of template" and " Efficient detection of viral RNA"). The innovative, dual-cation PCR buffer provided with the kit ensures high yields of specific PCR product over a wide range of annealing temperatures (see figure " Influence of annealing temperature on specificity"). Suboptimal RT-PCR is improved using Q-Solution, a unique additive that facilitates reverse transcription and amplification of templates with a high GC content or a high degree of secondary structure (see figure " RT-PCR of GC-rich template").
Principle
The QIAGEN OneStep RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template.
The QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend for both reverse transcription and PCR. The unique combination of Omniscript and Sensiscript Reverse Transcriptases, with their high affinity for RNA templates, ensures highly efficient and sensitive transcription of RNA amounts from as little as 1 pg up to 2 µg. After reverse transcription, reactions are heated to 95°C for 15 minutes to activate HotStarTaq DNA Polymerase and simultaneously inactivate the reverse transcriptases. This hot-start step eliminates nonspecific amplification products such as primer dimers and reduces background smear, ensuring highly sensitive and reproducible RT-PCR (see figures "Highly specific RT-PCR using low amounts of template" and "Efficient detection of viral RNA").
Wide range of annealing temperatures
The optimal primer annealing temperature is dependent on the base composition (i.e., the proportion of A, T, G, and C nucleotides), primer concentration, and ionic reaction environment. QIAGEN PCR Buffers contain both K+ and NH4+ and deliver high yields of specific PCR product over a wide range of annealing temperatures (see figure "Increased specific primer annealing"). This specificity is achieved by destabilizing non-specifically bound primers, providing a more robust reaction environment and eliminating the need for tedious annealing temperature optimization. In contrast, the range of optimal PCR annealing temperatures is smaller and less predictable using a PCR or one-step RT-PCR buffer that only contains K+, as illustrated in figure "Influence of annealing temperature on specificity".
QIAGEN OneStep RT-PCR Buffer has been specially developed to allow both efficient reverse transcription and PCR amplification. The buffer contains novel additives that prevent inhibition of PCR amplification by reverse transcriptases, a problem often encountered in one-step RT-PCR. The buffer ensures specific primer annealing over a wide range of temperatures and Mg2+ concentrations; providing robust and highly efficient RT-PCR from any RNA template. The QIAGEN OneStep RT-PCR Kit includes Q-Solution, an innovative additive that modifies the melting behavior of nucleic acids. Q-Solution facilitates reverse transcription and amplification of templates with a high GC content or a high degree of secondary structure (see figure "RT-PCR of GC-rich template"). Using Q-Solution simplifies optimization of RT-PCR for difficult templates. The QIAGEN OneStep RT-PCR Kit includes everything required for faster and easier RT-PCR for even the most sensitive applications (see table).
| HotStarTaq DNA Polymerase |
Highly specific products |
| Sensiscript and Omniscript Reverse Transcriptases |
Wide range of RNA amounts (1 pg – 2 µg) High sensitivity |
| OneStep RT-PCR Buffer |
Minimal optimization needed No inhibition of PCR by reverse transcriptases Inhibition of RNases |
| Q-Solution |
Facilitates amplification of GC-rich templates |
Procedure
The QIAGEN OneStep RT-PCR Kit allows fast and easy RT-PCR setup. Whatever the application — virus detection, molecular diagnostics research, or gene expression analysis — just mix all components together in one tube and start the thermal-cycler program (see flowchart "OneStep RT-PCR procedure"). The reaction mixture contains all of the reagents required for both reverse transcription and PCR; nothing needs to be added once the reaction has been started (see table).
Applications
The QIAGEN OneStep RT-PCR Kit is suitable for RT-PCR applications such as:
- Virus detection
- Single-cell RT-PCR
- Gene-expression analysis
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For fast and efficient one-step RT-PCR
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OneStep RT-PCR实验方案。
QIAGEN OneStep RT-PCR Kit可实现快速简单的RT-PCR体系构建。不论是哪种应用领域——病毒检测、分子诊断研究或基因表达分析——只需将所有组分在一个管内混匀并启动热循环仪程序即可。反应混合物包含进行逆转录和PCR所需的所有试剂;反应开始后无需再加入任何试剂。
使用少量模板进行高度特异性的RT-PCR。
使用图示量的HeLa细胞总RNA及α-连环蛋白特异性引物进行一步法RT-PCR,扩增690 bp的产物。所有反应均遵循供应商的说明进行。M:100 bp分子量标准。
高效检测病毒RNA。
对持续感染的Vero细胞中提取的Sendai病毒RNA进行逆转录并扩增336 bp的F-基因mRNA片段。使用QIAGEN OneStep RT-PCR Kit和图示量的病毒基因组拷贝进行反应。M:分子量标准。(数据由德国Martinsried的Max Planck Institute for Biochemistry的H. Rausch提供,数据是巴伐利亚州环境部支持的“安全等级2级和3级的病毒操作的实验控制”计划的一部分。)
退火温度对特异性的影响。
使用指定供应商提供的试剂盒,在一定的退火温度范围内进行一步法RT-PCR。从Hela RNA (箭头) 中逆转录并扩增1289 bp的人RCC1基因片段。M:分子量标准。仅使用QIAGEN OneStep RT-PCR Kit即可实现高特异性扩增,且无需优化。
对富含GC的模板进行RT-PCR。
从HeLa细胞总RNA中逆转录并扩增439 bp的转录因子TAFII100 mRNA片段 (GC含量:75%)。使用QIAGEN OneStep RT-PCR Kit,加入 (+) 或不加 (–) Q-Solution,进行反应。Q-Solution可确保富含GC的模板的特异性扩增。M:分子量标准。
退火时引物结合的特异性增强。
QIAGEN PCR缓冲液中的铵态氮和钾离子能够在退火时促进引物的特异性结合。K+结合到DNA主链上的磷酸基团(P–)上,稳定结合到模板上的引物。在热循环中,NH4+以铵离子和氨的形式存在,可以与碱基(B)之间的氢键发生相互作用,使错配碱基间的不稳定的氢键容易断开。两种阳离子的共同作用,在广泛的温度范围内保持特异性结合比例高。
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