Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Quantification of HIV-1 DNA

HIV-1 DNA的量化

FR Ferdinand Roesch
OS Olivier Schwartz

发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.417 浏览次数: 12738

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Jul 2012

Advertisement

Technologies for RNA Detection
Special Issue on Protocols for Organoid Research
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

精浆中HIV RNA 和人疱疹病毒DNA的量化

精浆中HIV RNA 和人疱疹病毒DNA的量化

Milenka V. Vargas-Meneses [...] Sara Gianella

2015年05月05日 11440 阅读

人肝细胞的HBV感染和壳体化HBV DNA的量化

人肝细胞的HBV感染和壳体化HBV DNA的量化

Kai Li [...] Akinori Takaoka

2016年01月20日 11377 阅读

全血HIV-2 DNA的定量分析

全血HIV-2 DNA的定量分析

Zsófia Szojka [...] Patrik Medstrand

2019年10月20日 6322 阅读

Abstract

The reverse transcription (RT) reaction is a critical step in HIV-1 life cycle. It is very strongly regulated and the target of several restriction factors (TRIM5α, APOBECs, SAMHD1, etc.). The progress of reverse transcription can be followed by measuring viral DNA by quantitative PCR (qPCR). This method is sensitive enough to allow detection of low amounts of HIV-1 DNA in infected cells and discriminate between several types of reverse transcription intermediates (so called 《early》 and 《late》 RT products, 2 Long Terminal Repeat (LTR) circles, integrated DNA).

Materials and Reagents

  1. Cells of interest, infected with HIV-1
  2. Proteinase K (20 mg/ml) (> 30 U/mg) (Eurobio, catalog number: GEXPRK01-B5 )
  3. AL buffer (QIAEN, catalog number: 19075 )
  4. Phenol-chloroform – isoamyl alcohol (Sigma-Aldrich, catalog number: 77617-100 ml )
  5. Chloroform (Sigma-Aldrich)
  6. Pure water (Life Technologies, Gibco®)
  7. Absolute Ethanol (Sigma-Aldrich)
  8. 3 M sodium acetate (pH 5.0)
  9. DpnI Fast Digest restriction enzyme and reaction buffer (Fermentas, catalog number: FD1704 )
  10. Platinium SYBR Green qPCR Supermix UDG (Life Technologies, InvitrogenTM, catalog number: 11733-046 )
  11. qPCR primers
  12. Standard plasmids for HIV-1 (pNL4-3) and GAPDH (pGAPDH)

Equipment

  1. Centrifuge
  2. DNA-free hood
  3. Speed-vac
  4. Heat-seal machine
  5. qPCR machine 
  6. 96-well qPCR plates (Eppendorf)
  7. Plastic films for qPCR plates (Eppendorf)
  8. Filter tips

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Roesch, F. and Schwartz, O. (2013). Quantification of HIV-1 DNA. Bio-protocol 3(7): e417. DOI: 10.21769/BioProtoc.417.
  2. Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物遗传学 > DNA > DNA 定量

微生物学 > 微生物-宿主相互作用 > 病毒

分子生物学 > RNA > qRT-PCR

您对这篇实验方案有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件