在终点法PCR中,用于RNA模板量为50 ng到2 μg的逆转录反应

  • 高亲和力酶获得高产量cDNA
  • 灵敏的检测低至10拷贝的模板
  • 无需额外的RNase H消化
  • 快速简单的操作流程,无需繁琐的移液步骤

Omniscript Reverse Transcription (RT) Kit专为单次逆转录反应中使用50 ng至2 μg RNA模板而设计。与RNA的高亲和力使得Omniscript Reverse Transcriptase比其它逆转录酶表现更出色,即使是低拷贝模板,在RT-PCR中也能实现更高的灵敏度。Omniscript RT Kit包括Omniscript Reverse Transcriptase、dNTPs和优化的反应缓冲液。只需加入引物,即可简单快速合成cDNA。

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Omniscript RT Kit (50)
For 50 reverse-transcription reactions: 200 units Omniscript Reverse Transcriptase, 150 µl 10x Buffer RT, 100 µl dNTP Mix (contains 5 mM each dNTP), 1.1 ml RNase-free water
205111
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Omniscript RT Kit (200)
For 200 reverse-transcription reactions: 800 units Omniscript Reverse Transcriptase, 4 x 150 µl 10x Buffer RT, 4 x 100 µl dNTP Mix (contains 5 mM each dNTP), 4 x 1.1 ml RNase-free water
205113
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The Omniscript RT 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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Full-length RT-PCR products — A.|Full-length RT-PCR products — B.|Omniscript Reverse Transcriptase procedure.|Comparison of various reverse transcriptases.|Sensitive RT-PCR of ≥10 copies.|
RT-PCR was carried out with total RNA from maize leaves using the indicated RT reaction temperatures. A 1.2 kb fragment of the GC-rich maize gl2 transcript was amplified. RT was carried out using the standard Omniscript RT protocol at the standard (37°C) and higher reaction temperatures as indicated.
|RT-PCR was carried out with total RNA from maize leaves using the indicated RT reaction temperatures. A 1.2 kb fragment of the GC-rich maize gl2 transcript was amplified. RT was carried out using the standard MMLV RNase H- RT protocol (Supplier L) at the standard (42°C) and other reaction temperatures, as indicated. This standard protocol was carried out with the required preliminary denaturation step and the recommended RNase H digest step following RT.
|The Omniscript RT Kit includes dNTPs and an optimized reaction buffer that, together with the high affinity of Omniscript Reverse Transcriptase for RNA, enables read-through of templates with high GC content or complex secondary structures. Due to this pre-optimization, tedious pipetting and pre-incubation steps are eliminated, and no additional RNase H digestion step is required.|Reverse transcription was carried out with different reverse transcriptases according to suppliers' specifications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplification with QIAGEN Taq DNA Polymerase. A 1.7 kb beta-actin fragment was amplified. Omniscript: Omniscript Reverse Transcriptase (QIAGEN); MMLV RNase H–: RNase H– reverse transcriptase from Moloney murine leukemia virus (Supplier L); MMLV: wild-type MMLV reverse transcriptase (Supplier L); AMV RNase H–: thermostable RNase H– reverse transcriptase from avian myeloblastosis virus (Supplier L); AMV: wild-type AMV reverse transcriptase (Supplier P).|A 700 nt in vitro transcript was reverse transcribed from 10 to 104 copies of starting template (in 200 ng total RNA), followed by PCR. 1/4 of each reverse-transcription reaction (corresponding to 2.5 to 2500 cDNA copies) was used for PCR amplification of a 330 bp fragment.|
Performance

高亲和力Omniscript Reverse Transcriptase (RT)在RT-PCR中具有高特异性和高灵敏度,即使是低拷贝转录(参见"Sensitive RT-PCR of ≥10 copies"),无需调整温度或反应条件即可通读复杂的RNA二级结构(参见"Comparison of various reverse transcriptases")。

GC含量高的RNA区域可能导致逆转录反应停止、从RNA模板脱离或跳过RNA环外区域(参见"Full-length RT-PCR products — B")。经证明,这些难扩增模板对QIAGEN逆转录酶没有影响(参见"Full-length RT-PCR products — A")。无需优化,Omniscript RT Kit就可使逆转录反应顺利进行。

Principle

Omniscript Reverse Transcriptase (RT)与RNA的高亲和力确保多种模板逆转录反应的高效性和灵敏性,获得高产量cDNA。提供即用型dNTPs,经优化的反应缓冲液和与RNA高亲和力的Omniscript RT,可通读GC含量高或复杂二级结构的模板。请注意,不提供引物混合物。

Omniscript RT专为逆转录反应设计,单次反应使用50 ng到2 µg的RNA模板。另可作为病毒RNA的酶选择,因为在多数病毒RNA制备中存在载体RNA。在对比实验中,Omniscript RT的表现在一系列不同RNA起始量中都优于其它逆转录酶。

QIAGEN提供严格的质量控制,确保Omniscript Kits批与批之间的高重复性。所有Omniscript RT Kits都包括经优化的Buffer RT、dNTPs和纯水,保证不含RNase、每批Omniscript RT都进行全面得RT-PCR重复性测试。

Procedure
使用经优化的Omniscript Reverse Transcriptase反应缓冲液,无需繁琐的移液和预培养步骤,无需另外使用RNase H消化步骤(参见"Omniscript Reverse Transcriptase Procedure")。
Applications

Omniscript Reverse Transcriptase适合以下应用:

  • 合成cDNA ,用于终点式PCR
  • 合成双链cDNA,用于克隆
  • RACE(cDNA终点的快速扩增)
  • 线性RNA扩增
  • 指数型RNA扩增
  • SAGE(基因表达序列分析)
  • 微阵列标记
  • 引物延伸转录起始位点分析
Feature
Specifications
Applications RT-PCR, qRT-PCR, primer-extension, RACE analysis
Enzyme activity Reverse transcription
Mastermix No
Reaction type Reverse transcription
Real-time or endpoint Endpoint
Sample/target type RNA template
Single or multiplex Single
With/without hotstart Without hotstart

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Kit Handbooks
1
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR
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