QIAGEN OneStep Ahead RT-PCR Kit

适用于快速一步法RT-PCR,灵敏度高、特异性强、保真性出色
  • 具有独特的两阶段热启动特性,确保可在室温条件下进行反应体系构建
  • 专有设计的酶混合液可显著提高反应特异性、灵敏度和保真性
  • 速度更快的1小时快速热循环程序
  • 内置加样变色指示剂,减少移液错误
  • 双重PCR扩增能力,可同时扩增内对照或内参
QIAGEN One-Step Ahead RT-PCR Kit中含有Sensiscript和Omniscript两种不同的逆转录酶,以及平衡配比的Taq DNA聚合酶和具有校对功能的高保真酶。热启动酶使得反应体系构建得以在室温条件下进行,便利的单管操作和优化的反应体系确保实验获得高灵敏度结果。带有颜色的指示剂染料使得实验人员能够分别对于电泳过程进行监控,并减少加样过程的失误。
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QIAGEN OneStep Ahead RT-PCR Kit (50)
6 vials for 50 reactions: 1 x 500 µl OneStep Ahead RT-PCR Master Mix, 1 x 50 µl OneStep Ahead RT Mix, 1 x 200 µl Template Tracer, 1 x 50 µl Master Mix Tracer, 1 x 1.9 ml water, 1 x 400 µl Q-Solution
220211
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QIAGEN OneStep Ahead RT-PCR Kit (200)
8 vials for 200 reactions: 2 x 1 ml OneStep Ahead RT-PCR Master Mix, 1 x 200 µl OneStep Ahead RT Mix, 1 x 200 µl Template Tracer, 1 x 50 µl Master Mix Tracer, 2 x 1.9 ml water, 1 x 2 ml Q-Solution
220213
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QIAGEN OneStep Ahead RT-PCR Kit (2000)
75 vials for 2000 x 25 µl reactions: 20 x 1 ml OneStep Ahead RT-PCR Master Mix, 10 x 200 µl OneStep Ahead RT Mix, 10 x 200 µl Template Tracer, 10 x 50 µl Master Mix Tracer, 20 x 1.9 ml water, 5 x 2 ml Q-Solution
220216
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Superior stability after reaction setup at room temperature|Amplification of GC-rich amplicons|Amplification of long amplicons|NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing|Optional pipetting control|Superior sensitivity|Superior specificity|OneStep Ahead RT-PCR procedure|
HeLa total RNA (10 and 1 pg) was used as a template for amplification of ACTB in triplicates, according to suppliers’ instructions. Reactions were either set up on ice or left at room temperature for the times indicated before analysis on a 2% agarose gel. Distinct, gene-specific bands are observed with the QIAGEN OneStep Ahead RT-PCR Kit even after a 2 h incubation at room temperature before cycling (blue arrow), whereas reactions performed with kits from competitors deteriorate as time progresses. Gene-specific bands appear significantly weaker, if present at all, while primer-dimers (red arrows) become more prominent.|HeLa total RNA (100 ng) was used as template for amplification of TNFRI (581 bp) with a GC ratio of 67.1%. Reactions were performed in triplicate, according to the suppliers’ instructions. Green arrows indicate specific product. Q-Solution was used for the QIAGEN reactions.|HeLa total RNA (100 and 10 ng) was used as a template for amplification of RANBP2 (3 kbp).|Potassium ions (K+) bind to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as ammonium ion and as ammonia under thermal‐cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing 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.|The kit comes with optional pipetting controls. The Master Mix tracer is an inert orange dye that can be added to the Master Mix. The Template Tracer is an inert blue dye that can be added to the template. When adding the template to the Master Mix, the color turns green, providing a visual indication of correct pipetting. In addition, both dyes allow tracking during gel electrophoresis. The dyes run at about 50 bp (orange) and 4000 bp (blue) on a 1% agarose gel.|Indicated amounts of HeLa total RNA (in pg) were used as template for amplification of GAPDH (831 bp) and ACTB (295 bp) in duplicate, according to the suppliers’ instructions. Green arrows indicate specific product, red arrows indicate primer‐dimers. Analysis was performed using the QIAxcel.|Indicated amounts of HeLa total RNA (in ng) were used as template for amplification of EIF2B4 (107 bp) in duplicate, according to the suppliers’ instructions. Green arrows indicate specific product, red arrows indicate primer‐dimers, purple arrows indicate nonspecific amplification products. Analysis was performed using the QIAxcel.|The QIAGEN OneStep Ahead RT-PCR Kit allows fast and easy RT-PCR setup whatever your application - virus detection, molecular diagnostics research or gene expression - just mix all components together in one tube and start your 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. As a visual pipetting control, you can add the optional orange Master Mix Tracer and blue Template Tracer.|
Performance
QIAGEN OneStep Ahead RT-PCR Kit可对任意RNA模板进行便利、高灵敏度、特异性的RT-PCR。该试剂盒含有经优化的组分,使得逆转录和PCR扩增可在同一混合液中“一步”完成。独特的酶配比和专门的反应缓冲液确保了高效、特异性的逆转录反应和PCR反应在同一反应管中有序进行,无需额外的优化步骤(参见图示Superior sensitivity以及Superior specificity)。

​在逆转录步骤,Omniscript和Sensiscript逆转录酶上结合有逆转录阻断基团,使其处于非激活状态,不会在室温条件下生成非特异的转录产物。当温度升高至50°C时,阻断基团从逆转录酶上解离,逆转录酶迅速达到完全激活状态。

​PCR扩增步骤受三种经平衡配比的DNA聚合酶催化。在室温下,QuantiNova DNA Polymerase处于抗体介导的阻断酶活状态。它在95ºC孵育5分钟后即可恢复活性,在第一个热循环即可实现高度特异性的扩增。HotStarTaq DNA Polymerase的酶活性逐渐提高,确保PCR的高产量。具有校对功能的高保真DNA聚合酶具有3'→5'外切酶活性,同样是95ºC孵育5分钟活化,确保扩增的高度准确性和持续合成能力。预混液中含有新型双阳离子PCR buffer,确保在宽泛的退火温度范围内,都可生成高特异性PCR产物。RT-PCR反应还采用了Q-Solution,这一组分有助于对于富含GC或含有复杂二级结构的模板进行高效的逆转录和扩增(参见图示Amplification of GC‐rich amplicons)。

​室温下进行反应体系构建这一特点,使该试剂盒可便利的用于自动化体系构建流程中。PCR预混液含有反应所需缓冲液、dNTP和DNA聚合酶,而含有逆转录酶和RNase抑制剂的RT-Mix则单独包装,确保逆转录反应不会提前进行。在进行反应体系构建时,仅需将引物、模板RNA和RT-Mix加入预混液中。

​该试剂盒中含有加样指示剂,能有效防止移液错误的发生,确保结果的可靠。显色体系由预混液指示剂和模板指示剂两部分组成,通过颜色变化即可轻松判断反应体系中是否加入模板,减少反应构建时的失误(参见图示Optional pipetting control)。该试剂盒还经专门优化,可适用于双重PCR,能够在单一反应体系中同时扩增阳性内质控。试剂盒中含有的RNase抑制剂能够防止因RNase污染而导致的RNA降解。
Principle
QIAGEN OneStep Ahead RT-PCR Kit适用于对各种RNA模板进行灵敏、便利的一步法RT-PCR。酶混合液经过特别配比,适用用于逆转录和PCR扩增反应。Omniscript和Sensiscript逆转录酶对RNA模板具有高亲和性,即便对于低至1 pg的RNA也能进行高效、灵敏的逆转录。逆转录反应结束后,95°C孵育5分钟,激活DNA聚合酶,同时逆转录酶失活。这一热启动步骤能够避免产生引物二聚体等非特异性扩增产物,并且减少背景条带弥散的发生,确保高度灵敏性、可重复的RT-PCR反应。

​引物最佳退火温度取决于其碱基组成、引物浓度和离子反应环境。QIAGEN PCR Buffers含有K+和NH4+,确保在宽泛的退火温度范围内,均可生成特异性PCR产物(参见图示NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing)。在此条件下非特异性结合的引物二聚体无法稳定存在,因此可确保反应的特异性。由于反应对于温度条件的要求降低,减少了繁琐的温度优化过程。与此相反,在使用仅含有K+的的PCR或一步法RT-PCR缓冲液时,理想的PCR退火温度范围较小,并且难以预测。QIAGEN OneStep Ahead RT-PCR Buffer经特别设计,能够用于高效的逆转录和PCR扩增反应。缓冲液中含有的新型添加物组分能够防止逆转录酶对于PCR扩增反应的抑制,解决了这一在一步法RT-PCR经常面临的问题。这一特制的缓冲液确保引物能够在宽泛的温度和Mg2+浓度范围内实现与模板的特异性结合,进而确保对各种RNA模板进行灵敏、高效的RT-PCR反应。

QIAGEN OneStep Ahead RT-PCR Kit中含有特殊的Q-Solution组分,它能够对于核酸的熔解行为进行修饰,促进富含GC或二级结构的困难模板的逆转录和扩增。QIAGEN OneStep RT-PCR Kit含有进行RT-PCR的全部所需试剂,适用于即便是最敏感的应用(参见下表)。

可靠的一步法RT-PCR结果
QIAGEN OneStep Ahead RT-PCR 特性 优势
Sensiscript和Omniscript逆转录酶 宽泛的RNA模板量范围(0.1 pg – 2 µg)
灵敏性高
热启动活性
在室温条件下进行反应体系构建
可在自动化流程中使用
QuantiNova HotStarTaq热启动高保真聚合酶 特异性产物
热启动活性
更高的保真性
热循环时间短
便利的室温下反应体系构建
准确性高
可扩增长序列(<4 kb)
热循环时间短(约1小时)
含有DNA聚合酶、缓冲液和dNTP的预混液 专有的双阳离子缓冲液体系
经优化可用于双重PCR反应
无需优化
便利的反应体系构建
逆转录酶不会抑制PCR反应
可同时扩增阳性对照,提高反应可靠性
RNase抑制剂 抑制RNase活性 避免RNA降解
Q-Solution 促进富含GC的模板扩增 反应性能更加可靠
加样变色指示剂 惰性的橙色和蓝色染料
凝胶电泳时橙色染料为50 bp,蓝色为4000 bp
减少移液错误,确保反应准确性
可作为凝胶示踪染料


Procedure
QIAGEN OneStep Ahead RT-PCR Kit能够进行便利、快速的RT-PCR反应,适用于多种应用,包括病毒检测、分子诊断研究、以及基因表达分析。只需将逆转录酶混合液、引物和模板加入到预混液中,即可开始热循环。反应体系构建可以在室温条件下进行,可选择性加入预混液和模板变色指示剂,以避免在移液过程中可能发生的错误。反应混合液中含有逆转录和PCR扩增反应所需的全部试剂。
Applications
QIAGEN OneStep Ahead RT-PCR Kit适用于以下RT-PCR应用:
  • 病毒检测   
  • 基因表达分析
Feature
Specifications
Applications Gene expression analysis, virus detection
Enzyme activity Reverse transcription, 5' -> 3' exonuclease activity
Mastermix Yes
Reaction type One-step RT-PCR
Real-time or endpoint End-point
Sample/target type RNA template
Single or multiplex Single/duplex
With/without hotstart With hot-start

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