用于灵敏、特异性的一步法RT-PCR

  • 快速方便的单管反应
  • 适合各种RNA模板,无需优化反应条件
  • 独特的酶组合,确保反应的高特异性和高灵敏度
  • 经优化的缓冲体系确保了高效的逆转录和扩增过程
QIAGEN One-Step RT-PCR Kit包含Omniscript逆转录酶和Sensiscript逆转录酶的混合物、HotStarTaq DNA Polymerase、QIAGEN OneStep RT-PCR Buffer、dNTP混合液和Q-Solution。Q-Solution是一种新型的辅助剂,有助于实现“困难”模板(如:高GC含量的模板)的高效扩增。简单的单管反应体系构建和优化的试剂盒组分确保了结果成功且灵敏度高。
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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)
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)
210212
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The QIAGEN OneStep RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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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
QIAGEN OneStep RT-PCR Kit提供方便的规格,可使用RNA模板进行高灵敏度和特异性的RT-PCR。试剂盒中包含优化的组分,可在同一反应混合液中进行逆转录和PCR扩增,完成"一步式"反应。独特的酶组合和专门研发的反应缓冲液确保在一个管中进行高效、高特异性的逆转录和PCR,无需优化(参见"Highly specific RT-PCR using low amounts of template" 和 "Efficient detection of viral RNA")。试剂盒提供的新型的、双阳离子PCR缓冲液确保在宽退火温度范围内得到高产量的特异性PCR产物(参见"Influence of annealing temperature on specificity")。使用Q-Solution可提高不理想的RT-PCR的表现,这种独特的辅助剂可帮助逆转录并高效扩增具有高GC含量和复杂二级结构的模板(参见"RT-PCR of GC-rich template")。
Principle

QIAGEN OneStep RT-PCR Kit可对多种RNA模板进行简单、灵敏的一步式RT-PCR。

QIAGEN OneStep RT-PCR Enzyme Mix含有为逆转录和PCR反应特别设计的酶。独特组合的Omniscript和Sensiscrip逆转录酶对RNA模板有高度亲和性,确保RNA逆转录的灵敏度达到1 pg到2 µg。逆转录完成后,升温至95°C,15分钟,活化HotStarTaq DNA Polymerase同时使逆转录酶失活。这种方法可减少引物二聚体等非特异性扩增产物的生成,并减少背景的影响,确保RT-PCR的高灵敏度和可重复性。

退火温度范围宽

引物的最佳退火温度取决于碱基对的组成(如A、T、G、C核苷酸的比例)、引物的浓度和反应的离子环境。QIAGEN PCR Buffers含有K+和NH4+,使用该缓冲液可在很宽的温度范围内增加特异性PCR产物的产量(参见"Increased specific primer annealing")。其原理是破坏非特异性结合的引物对,提供更加可靠的反应环境,减少繁琐的温度优化过程。相反,如使用仅含K+的PCR或一步式RT-PCR缓冲液,PCR最佳退火温度的范围较窄,可靠性较差。

QIAGEN OneStep RT-PCR Buffer专为提高逆转录和PCR扩增的效率而设计。该缓冲液含有新型辅助剂,可防止逆转录酶对PCR扩增的抑制,而这是一步式RT-PCR中常见的问题。该缓冲液确保在较大的温度范围和Mg2+浓度范围内,引物与模板特异性结合,可对多种RNA模板进行高效的RT-PCR扩增。QIAGEN OneStep RT-PCR Kit含有Q-Solution,这是可修饰核酸熔解行为的新型辅助剂。Q-Solution可促进高GC含量或含较多二级结构的模板的逆转录和扩增。 使用Q-Solution可简化复杂模板RT-PCR的优化过程。QIAGEN OneStep RT-PCR Kit含有您所需的所有试剂,可进行快速、简便的RT-PCR,用于多种灵敏的应用(见表)。

可靠的一步式RT-PCR结果
QIAGEN OneStep RT-PCR Kit 组分 特点
HotStarTaq DNA Polymerase 高度特异性产物
Sensiscript and Omniscript Reverse Transcriptases RNA初始量的范围大 (1 pg–2 µg)
高灵敏度
OneStep RT-PCR Buffer 无需优化
反转录酶不会抑制PCR
抑制RNases
Q-Solution 促进GC含量高的模板的扩增

 

Procedure

使用QIAGEN OneStep RT-PCR Kit可快速、简单的进行RT-PCR反应体系构建。无论是何种应用,即无论是病毒检测、分子诊断研究还是基因表达,您只需将所有试剂在一个离心管中混合即可开始PCR扩增(参见"OneStep RT-PCR procedure")。反应混合液含有逆转录和PCR反应所需的所有试剂,无需添加其他试剂即可开始反应(参见下表)。

Applications

QIAGEN OneStep RT-PCR Kit适用于RT-PCR分析,包括:

  • 病毒检测
  • 单细胞RT-PCR
  • 基因表达分析
Feature
Specifications
Applications Gene-expression analysis, virus detection
Enzyme activity Reverse transcription, 5' -> 3' exonuclease activity
Mastermix No
Reaction type One-step RT-PCR
Real-time or endpoint Endpoint
Sample/target type RNA template
Single or multiplex Single
With/without hotstart With hotstart

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Kit Handbooks
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References
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