Polysome Preparation, RNA Isolation and Analysis

Materials and Reagents

1. Cells (Neuroblastoma cell line SKN-SH)
   
2. Protease inhibitors (Sigma-Aldrich, catalog number: P8340-5ML )
   
3. DTT (Sigma-Aldrich, catalog number: 43815 )
   
4. Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
   
5. Heparin (Sigma-Aldrich, catalog number: H3149 )
   
6. Sucrose (Sigma-Aldrich, catalog number: S1888 )
   
7. Phenol/chloroform/isoamyl alcohol (Life Technologies, Invitrogen™, catalog number: 15593-031 )
   
8. Sodium acetate (Thermo Fisher Scientific, catalog number: S209-500 )
   
9. 1x PBS
   
10. 1x Trypsin-EDTA, 0.05% Trypsin/0.53 mM EDTA (Cellgro, catalog number: 25-052-CV )
    
11. RPMI-1640 (Hyclone, catalog number: SH30096.01 )
    
12. Tris-Base (Thermo Fisher Scientific, catalog number: BP-152-1 )
    
13. KCl (Thermo Fisher Scientific, catalog number: BP-366-500 )
    
14. MgCl₂ (Sigma-Aldrich, catalog number: M-2393 )
    
15. Triton X-100 (Sigma-Aldrich, catalog number: T8787-250ML )
    
16. DNAase/RNAase free Ethanol (Sigma-Aldrich, catalog number: E7023-500ML )
    
17. DNAase/RNAase free water (BioExpress, catalog number: UPW-1000 )
    
18. Ultracentrifuge tubes (14 x 89 mm) (Beckman Coulter, catalog number: 344059 )
    
19. Polysome extraction buffer (PEB) (see Recipes)
    
20. Sucrose solutions (see Recipes)
    

Equipment

1. BR-188 Density gradient fractionation system (Brandel)
   
2. Beckman optima L-70 ultracentrifuge (Beckman)
   
3. 7500 Real-time PCR system (Applied Biosystems)
   
4. Boekel scientific orbitron rotator I, 115 V (Boekel Scientific)
   

Procedure