Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR
通过量化分析RT-qPCR将逆转录病毒RNA整合到病毒颗粒中
发布: 2013年04月20日第3卷第8期 DOI: 10.21769/BioProtoc.684 浏览次数: 16640
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
Abstract
Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation efficiency (Brandt et al., 2007; Blissenbach et al., 2010; Grewe et al., 2012).
Keywords: HIV (艾滋病咨询门诊)
Encapsidation (包装)
Packaging (包装)
RT-PCR (RT-PCR)
Viral Particles (病毒颗粒)
Materials and Reagents
I. Cell harvesting and ultracentrifugation
- Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 )
- Fetal calf serum (FBS) (Life Technologies, Gibco®, catalog number: 10108-165 )
- Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14200-067 )
- Trypsin (Biochrom)
- Penicillin/Streptomycin (Life Technologies, Gibco®)
- 0.45 μm filter (Sarstedt)
- Sucrose (Biomol)
- Ultracentrifugation tubes (17 ml) (Herolab)
II. Cell fractionation and RNA isolation
- Dithiothreitol (DTT) solution (1 M) (AppliChem GmbH, catalog number: A3668,0050 )
- RiboLock RNase Inhibitor (40 U/μl) (Thermo Fisher Scientific, catalog number: E00384 )
- Ethanol >99.8% (Sigma-Aldrich)
- QIAshredder Spin Columns (QIAGEN, catalog number: 79654 )
- RNeasy Kit (QIAGEN, catalog number: 74106 )
- QIAmp Viral RNA Kit (QIAGEN, catalog number: 52906 )
- TURBO DNA-free Kit (Life Technologies, Ambion®, catalog number: AM1907 )
- Nuclease-free water (B Braun)
- Quant-iT RNA Assay Kit (Life Technologies, InvitrogenTM, catalog number: Q-33140 )
III. RT-qPCR
- QuantiTect Probe RT-PCR Kit (includes Reverse Transcriptase) (QIAGEN, catalog number: 204443 )
- SYBR Green (Life Technologies, InvitrogenTM)
- Primers sense and antisense (Biomers)
- Isolated RNA
- TOPO TA Cloning Kit (Life Technologies, InvitrogenTM, catalog number: 45-0640 )
- AmpliScribe-T7/Sp6 High Yield Transcription Kit (Epicentre, catalog number: AS2607 )
- RNA standards
IV. Other materials
- HIV-1 DIY p24 sandwich ELISA Kit 2 (Aalto Bio Reagents)
Equipment
- Heraeus Multifuge X3R Centrifuge (Thermo Fisher Scientific)
- Centrifuge 5417R (Eppendorf)
- Sorvall WX Ultra 100 Ultracentrifuge (Thermo Fisher Scientific)
- Qubit Fluorometer (Life Technologies, InvitrogenTM, catalog number: Q32857 )
- Rotor-Gene Q (QIAGEN, catalog number: 9001620 )
- Strip tubes 0.1 ml (QIAGEN)
- 15 ml tubes (Sarstedt)
- 1.5 ml tubes (Sarstedt)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hoffmann, B. and Grewe, B. (2013). Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR. Bio-protocol 3(8): e684. DOI: 10.21769/BioProtoc.684.
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分类
微生物学 > 微生物遗传学 > RNA
分子生物学 > RNA > qRT-PCR
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