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Biology
牛痘病毒感染及病毒基因表达的时空分析:第2部分
牛痘病毒感染及病毒基因表达的时空分析:第2部分
JoVE Journal
Biology
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JoVE Journal Biology
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

牛痘病毒感染及病毒基因表达的时空分析:第2部分

Full Text
8,362 Views
10:55 min
April 10, 2009

DOI: 10.3791/1169-v

Judy Yen1, Ron Golan1, Kathleen Rubins1

1Whitehead Institute for Biomedical Research,MIT - Massachusetts Institute of Technology

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This protocol outlines the process for vaccinia virus infection of HeLa cells and subsequent analysis of both host and viral gene expression. The focus is on synthesizing cDNA from isolated RNA for further analysis.

Key Study Components

Area of Science

  • Virology
  • Cell Biology
  • Gene Expression Analysis

Background

  • Vaccinia virus is a model organism for studying viral infections.
  • HeLa cells are widely used in biological research.
  • Understanding host and viral gene expression is crucial for virology.
  • Microarray analysis allows for comprehensive gene expression profiling.

Purpose of Study

  • To investigate the gene expression changes in HeLa cells post-infection.
  • To isolate and amplify viral RNA for analysis.
  • To provide a detailed protocol for researchers studying viral infections.

Methods Used

  • Infection of HeLa cells with vaccinia virus.
  • Isolation of total RNA from infected cells.
  • Reverse transcription to synthesize cDNA from RNA.
  • Preparation for microarray analysis of gene expression.

Main Results

  • Successful isolation of total RNA from infected HeLa cells.
  • Amplification of viral RNA using reverse transcription.
  • Preparation of samples for subsequent microarray analysis.
  • Insights into host and viral gene expression dynamics.

Conclusions

  • The protocol provides a reliable method for studying viral infections.
  • Amplified RNA can be effectively used for microarray analysis.
  • Understanding gene expression changes can inform therapeutic strategies.

Frequently Asked Questions

What is the significance of using HeLa cells?
HeLa cells are a standard model for studying human cell biology and are particularly useful for viral infection studies.
How is RNA isolated from infected cells?
RNA is isolated using standard extraction protocols that separate RNA from cellular components.
What is the role of reverse transcription in this protocol?
Reverse transcription converts RNA into cDNA, which is necessary for amplification and analysis.
What type of analysis is performed after RNA amplification?
Microarray analysis is performed to assess the expression levels of host and viral genes.
Can this protocol be applied to other cell types?
Yes, while this protocol uses HeLa cells, it can be adapted for other cell types as well.

协议牛痘感染的HeLa细胞和宿主和病毒基因的表达分析。

大家好,我是来自怀特黑德生物医学研究所 Kate Rubins 实验室的 Judy Yen。用牛痘病毒感染 hela 细胞并分离总 RNA 后,如之前的 Joe 视频方案帖子所示,可以使用反向和体外转录分离和扩增病毒 RNA,扩增的 RNA 稍后将用于病毒和宿主基因表达的微阵列分析。那么让我们开始吧。

为了开始从 RNA 合成 c cd NA,在洗涤缓冲液中使用环境氨基等位基因信息 AMP two 试剂盒之前,将推荐体积的 100% 乙醇加入洗涤缓冲液中,在 PCR 反应管中加入 100 ng 至 5 μg 总 RNA 和 1 μL T 7 寡核苷酸引物。用不含核酸酶的水将体积增加到 12 μL。将样品在 70 摄氏度下在热循环仪中孵育 10 分钟。

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细胞生物学 免疫学 微生物学 第26期 牛痘 病毒 感染 HeLa细胞 TRIzol试剂 总RNA 基因芯片 放大 氨基烯丙基 RNA的Ambion公司氨基烯丙基MessageAmpII 基因表达

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