QIAGEN OneStep RT-PCR Kit

用于灵敏、特异性的一步法RT-PCR

Features

  • 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

Products

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。
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QIAGEN OneStep RT-PCR Kit (100)

Cat. No. / ID: 210212

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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QIAGEN OneStep RT-PCR Kit (1000)

Cat. No. / ID: 210215

For 1000 x 50 µl reactions (available in a single tube): QIAGEN OneStep RT-PCR Enzyme Mix (2 x 1 ml), 5x QIAGEN OneStep RT-PCR Buffer (1 x 10 ml), dNTP Mix (2 x 1 ml, 10 mM each), 5x Q-Solution (1 x 10 ml), RNase-Free Water (1 x 40 ml)
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QIAGEN OneStep RT-PCR Kit (25)

Cat. No. / ID: 210210

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)

Product Details

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。

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")。
See figures

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

 

See figures

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)。

See figures

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

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsGene-expression analysis, virus detection
mastermixNo
enzymeactivityReverse transcription, 5' -> 3' exonuclease activity
reactiontypeOne-step RT-PCR
realtimeorendpointEndpoint
sampletargettypeRNA template
singleormultiplexSingle
withwithouthotstartWith hotstart

Publications

Up-regulation of cyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells.
Hinz B; Ramer R; Eichele K; Weinzierl U; Brune K;
Mol Pharmacol; 2004; 66 (6):1643-51 2004 Sep 10 PMID:15361550
Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA.
Kiehart DP; Franke JD; Chee MK; Montague RA; Chen TL; Roote J; Ashburner M;
Genetics; 2004; 168 (3):1337-52 2004 Nov PMID:15579689
Expression of growth differentiation factor 9 messenger RNA in porcine growing and preovulatory ovarian follicles.
Prochazka R; Nemcova L; Nagyova E; Kanka J;
Biol Reprod; 2004; 71 (4):1290-5 2004 Jun 9 PMID:15189836
Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus.
Lee CW; Senne DA; Suarez DL;
J Virol; 2004; 78 (15):8372-81 2004 Aug PMID:15254209

FAQ

Are there specific recommendations for performing RT-PCR on RNA isolated from paraffin-embedded samples?

Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure. 

If performing RT-PCR with degraded RNA, we recommend using gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation.

We recommend that the RNeasy FFPE or miRNeasy FFPE kit be used to isolate the RNA.

FAQ ID -828
Do I need to use an RNase inhibitor in my RT reaction?
To be on the safe side, we highly recommend the use of RNase inhibitors in all RT reactions, since RNases are nearly everywhere and it is very easy to contaminate samples during reaction setup. The reaction conditions used for RT are well-suited for RNase activity. Even traces of RNase can nick the RNA, causing shortened cDNA products, low yields, and reduced RT-PCR sensitivity.
FAQ ID -119
Is mRNA isolation necessary for sensitive RT-PCR?
Usually not. Since RT-PCR is extremely sensitive, as little as 10–200 ng total RNA is sufficient for each 25–50 µl reaction mix, depending on the RT system. For abundant mRNA species, it is possible to use even less than 10 ng total RNA. For rare mRNA species, use a sequence-specific primer in the RT reaction to increase sensitivity. RNA content in various cells and tissues can be found here.
FAQ ID -111
Does QIAGEN sell Q-Solution separately?
No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA PolymeraseQIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.
FAQ ID -204
Do you have a protocol for polyacrylamide gel analysis of oligonucleotides?
Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).
FAQ ID -961
Does the 5x OneStep RT-PCR Buffer contain BSA?
No. The 5x OneStep RT-PCR Buffer does not contain BSA.
FAQ ID -326
Can I shorten the activation time for the HotStarTaq DNA Polymerase?
No, the initial activation time of 15 minutes at 95°C is crucial. Enzyme activation will be incomplete when using shorter activation times, resulting in inefficient PCR product amplification.
FAQ ID -565