Polysome Preparation, RNA Isolation and Analysis
多核糖体的制备、RNA分离和分析
发布: 2012年11月05日第2卷第21期 DOI: 10.21769/BioProtoc.286 浏览次数: 27951
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参见作者原研究论文
The authors used this protocol in:
Mar 2012
Abstract
During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.
Materials and Reagents
- Cells (Neuroblastoma cell line SKN-SH)
- Protease inhibitors (Sigma-Aldrich, catalog number: P8340-5ML )
- DTT (Sigma-Aldrich, catalog number: 43815 )
- Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
- Heparin (Sigma-Aldrich, catalog number: H3149 )
- Sucrose (Sigma-Aldrich, catalog number: S1888 )
- Phenol/chloroform/isoamyl alcohol (Life Technologies, Invitrogen™, catalog number: 15593-031 )
- Sodium acetate (Thermo Fisher Scientific, catalog number: S209-500 )
- 1x PBS
- 1x Trypsin-EDTA, 0.05% Trypsin/0.53 mM EDTA (Cellgro, catalog number: 25-052-CV )
- RPMI-1640 (Hyclone, catalog number: SH30096.01 )
- Tris-Base (Thermo Fisher Scientific, catalog number: BP-152-1 )
- KCl (Thermo Fisher Scientific, catalog number: BP-366-500 )
- MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787-250ML )
- DNAase/RNAase free Ethanol (Sigma-Aldrich, catalog number: E7023-500ML )
- DNAase/RNAase free water (BioExpress, catalog number: UPW-1000 )
- Ultracentrifuge tubes (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
- Polysome extraction buffer (PEB) (see Recipes)
- Sucrose solutions (see Recipes)
Equipment
- BR-188 Density gradient fractionation system (Brandel)
- Beckman optima L-70 ultracentrifuge (Beckman)
- 7500 Real-time PCR system (Applied Biosystems)
- Boekel scientific orbitron rotator I, 115 V (Boekel Scientific)
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zhang, H. and Zhou, M. (2012). Polysome Preparation, RNA Isolation and Analysis. Bio-protocol 2(21): e286. DOI: 10.21769/BioProtoc.286.
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分类
分子生物学 > RNA > mRNA 转译
分子生物学 > RNA > RNA 提取
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