Omniscript RT Kit

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。

Product Cat. no. List price:
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
2 465,00 kr
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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
8 310,00 kr
Add to cart
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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全长的RT-PCR产物— A。|全长的RT-PCR产物— B。|Omniscript Reverse Transcriptase实验流程。|多种逆转录酶的对比。|对10个以上拷贝进行灵敏的RT-PCR。|
采用图示的RT反应温度,对从玉米叶中提取的总RNA进行RT-PCR。扩增1.2 kb的富含GC的玉米gl2转录本片段。采用标准的Omniscript RT实验方案,按说明在标准(37°C)和较高的反应温度下进行RT。|采用图示的RT反应温度,对从玉米叶中提取的总RNA进行RT-PCR。扩增1.2 kb的富含GC的玉米gl2转录本片段。采用标准的MMLV RNase H- RT实验方案(Supplier L),按说明在标准(42°C)及其他反应温度下进行RT。该标准实验方案包括必需的初步变性步骤以及在RT结束后推荐进行的RNase H酶酶切步骤。|Omniscript RT Kit包括dNTPs和优化的反应溶液以及对RNA高亲和性的Omniscript Reverse Transcriptase,能够通读高GC含量和二级结构复杂的序列。由于这种预优化,无需繁琐的移液和预孵育步骤,也无需额外的RNase H消化步骤。|使用从HeLa细胞中提取的图示量的总RNA,依照供应商提供的说明书使用不同的逆转录酶进行逆转录。1/20的逆转录反应使用QIAGEN Taq DNA Polymerase进行25个循环的PCR扩增反应。扩增1.7 kb的β-actin片段。Omniscrip:Omniscript Reverse Transcriptase(QIAGEN);MMLV RNase H–:来自Moloney小鼠白血病病毒的RNase H–逆转录酶(供应商L);MMLV:野生型MMLV逆转录酶(供应商L);AMV RNase H–:来自禽成髓细胞瘤病毒的RNase H–逆转录酶(供应商L);AMV:野生型AMV逆转录酶(供应商P)。|从10到104拷贝的起始模板中(在200 ng总RNA中)逆转录一个700 nt的体外转录子,随后进行PCR。每个逆转录反应的1/4(相当于2.5到2500拷贝的 cDNA)用于PCR扩增一个330 bp的片段。|
性能

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。

原理

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。

操作流程
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")。
应用

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

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Kit Handbooks
1
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR
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Safety Data Sheets
1
Download Safety Data Sheets for QIAGEN product components.
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Full-Length RT-PCR Products.
Full-length RT-PCR products — A.
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.
Full-Length RT-PCR Products.
Full-length RT-PCR products — B.
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.
Omniscript RT procedure
Omniscript Reverse Transcriptase procedure.

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.

Superior sensitivity and dynamic range of Omniscript RT
Comparison of various reverse transcriptases.
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).
Sensitive RT-PCR of ≥10 Copies
Sensitive RT-PCR of ≥10 copies.
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.