Isolation of Polysome-bound mRNA from Rice Solid Tissues Amenable for RT-PCR and Profiling Experiments
分离水稻固体组织的多核糖体结合mRNA用于RT-PCR和图谱实验
发布: 2015年03月05日第5卷第5期 DOI: 10.21769/BioProtoc.1411 浏览次数: 14945
评审: Ru ZhangAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2013
Abstract
Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.
Materials and Reagents
- 30-day-old rice roots or shoots
- Liquid nitrogen
- 3 M sodium acetate (Thermo Fisher Scientific, catalog number: S209-500 )
- Chloramphenicol (50 mg/ml) (Sigma-Aldrich, catalog number: C0378 )
- Cycloheximide (50 mg/ml) (Sigma-Aldrich, catalog number: C7698 ) in MilliQ water
Note: Cycloheximide inhibits protein synthesis by blocking translation elongation. This molecule interferes with the translocation of tRNAs with the mRNA and the ribosome, resulting in fixed ribosomes on mRNAs. - 8 M guanidine hydrochloride solution (Sigma-Aldrich, catalog number: G9284 )
- Chloroform
- Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
- Isopropanol
- KCl (Thermo Fisher Scientific, catalog number: BP-366-500 )
- Linear acrylamide (5 mg/ml) (Life Technologies, InvitrogenTM, catalog number: AM9520 )
- MgCl2 (Sigma-Aldrich, catalog number: M-2393 )
- Nonidet P40 (Biocompare, catalog number: DG514 )
- Phenol water saturated
- Sucrose (Sigma-Aldrich, catalog number: S1888 )
- Tris.HCl (pH 8.4) (Thermo Fisher Scientific, catalog number: BP152-1 )
- Trizol reagent (Life Technologies, InvitrogenTM, catalog number: 15596-026 )
- 10x sucrose solution salts buffer (see Recipes)
- 2 M sucrose (see Recipes)
- 20-50 % sucrose solutions (see Recipes)
- Polysome buffer (see Recipes)
Equipment
- 0.22 μm filter
- Density gradient fractionation system composed by
- Peristaltic pump (Gilson Minipuls 3 peristaltic pump)
- Cary 60 UV-Vis Spectrophotometer (Agilent)
- Flow cell quartz (1 mm 113 μl, 1/pk) (Agilent, catalog number: 6610019900 )
- Fraction collector (Bio-Rad Laboratories, model: 2110 )
- Peristaltic pump (Gilson Minipuls 3 peristaltic pump)
- Beckman optima L-70 ultracentrifuge (Beckman Coulter)
- Beckman tubes
- Parafilm
- Boekel scientific orbitron rotator I (115V) (Boekel Scientific)
- Mini centrifuge (Eppendorf, model: 5415D)
- NanoDrop (Thermo Fisher Scientific)
Software
- The Agilent Cary WinUV software
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q. and Poirier, Y. (2015). Isolation of Polysome-bound mRNA from Rice Solid Tissues Amenable for RT-PCR and Profiling Experiments. Bio-protocol 5(5): e1411. DOI: 10.21769/BioProtoc.1411.
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分类
植物科学 > 植物分子生物学 > RNA
分子生物学 > RNA > qRT-PCR
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