Polysome Analysis   

Materials and Reagents

1. Ultracentrifuge tube for SW41Ti (Seton Scientific, catalog number: 7030 )
   
2. Permanent ink marker
   
3. 10 ml syringe with wide gauge luer lock cannula (Vita Needles, gauge 14)
   
4. 10 cm dish (Eppendorf, catalog number: 0030702115 )
   
5. 1.5 ml tube (RNase and DNase free) (Corning, Axygen^®, catalog number: MCT-150-C or equivalent)
   
6. MCF7 human breast carcinoma cell line (ATCC, catalog number: HTB22 )
   
7. Cycloheximide (CHX) (AMRESCO, catalog number: 94271 )
   
8. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
   
9. DEPC treated water (AMRESCO, catalog number: E174 )
   
10. Citrate saturated phenol (pH 4.5) (Sigma-Aldrich, catalog number: P4682 )
    
11. Chloroform (Sigma-Aldrich, catalog number: C2432 )
    
12. 75% ethanol
    
13. Nuclease free water
    
14. Oligo dT primer
    
15. dNTP mix
    
16. 5x RT buffer
    
17. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: R0861 )
    
18. RNase inhibitor (Thermo Fisher Scientific, catalog number: EO0381 )
    
19. cDNA synthesis reagents (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 28025013 )
    
20. 10x PCR buffer (with 15 mM MgCl₂)
    
21. 10 µM forward primer for gene of interest/control gene
    
22. 10 µM reverse primer for gene of interest/control gene
    
23. PCR reagents (New England Biolabs, catalog number: M0273L )
    
24. Sucrose (RNase and DNase free) (AMRESCO, catalog number: 0335 )
    
25. Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
    
26. Magnesium chloride (MgCl₂) (AMRESCO, catalog number: 0288 )
    
27. HEPES (pH 7.4) (Thermo Fisher Scientific, Affymetrix, catalog number: 16926 )
    
28. IGEPAL CA-360 detergent (Sigma-Aldrich, catalog number: I3021 )
    
29. Protease inhibitor cocktail (AMRESCO, catalog number: M250 )
    
30. 10% sucrose solution (for 8 ml) (see Recipes)
    
31. 50% sucrose solution (for 8 ml) (see Recipes)
    
32. Polysome lysis buffer (see Recipes)
    

Equipment

1. Gradient Station or any equivalent gradient fractionators (Biocomp, catalog number: 153-002 )
   
2. Tube holder
   
3. Ultra-centrifuge (Beckman Coulter, model: Optima L-70 or equivalent)
   
4. Rotor SW41Ti (Beckman Coulter, catalog number: 331362 )
   
5. 200 µl pipette
   
6. Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: NanoDrop^TM 2000 )
   
7. Fraction collector (Gilson, model: FC203B )
   
8. UV monitor (Bio-Rad Laboratories, model: EM1-Econo^TM )
   
9. Balance
   
10. Vortex
    

Software

1. Gradient Master software (included with the Gradient Station)

Procedure

1. Gradient preparation
   1. Prepare 8 ml of 10% and 50% sucrose solution each in 1x sucrose gradient buffer for a single gradient (see Recipes, Table 1).
      
   2. Degas the solution under vacuum for 5 min.
      
   3. Mark ultracentrifuge tubes at 50% of tube height with permanent ink marker using the marker block provided with the Gradient Station.
      
   4. Carefully add 10% sucrose solution upto the 50% mark of the tube with a wide gauge cannula.
      
   5. Aspirate the 50% sucrose solution into a syringe and insert the cannula directly to the bottom of the tube through the 10% sucrose solution then slowly layer the heavier solution to the bottom of the tube without disturbing the 10% sucrose solution. The heavier sucrose solution will slowly push the 10% solution up, and the 50% sucrose solution should reach up to the 50% marking of the ultracentrifuge tube.
      
   6. Cover the tube with rubber cap and place the tube in the tube holder on the rotor of the gradient station.
      
   7. Run the following pre-set program in the Gradient Master software
      Program name: Short Sucrose 10-50% (w/v) Fast
      Program specification: 10-50%; Step: S1/1; Time: 1 min 45 sec; Angle: 81.5; rpm: 25 (Video 1).
      
   8. Carefully take out the tubes and store vertically at 4 °C for 2 h without disturbance.
      
      
      
      Video 1. Gradient preparation
      
      
2. Cell lysate preparation for polysome profiling
   
   1. Treat 70-80% confluent cells in a 10 cm dish with 100 µg/ml cycloheximide (CHX) by directly adding it to the culture media at 15 to 30 min prior to harvesting.
      
   2. Wash the cells twice with PBS containing 100 µg/ml CHX, and harvest them by scraping into 10 ml ice cold PBS containing 100 µg/ml CHX. Centrifuge at 600 x g for 5 min at 4 °C and discard the supernatant carefully.
      
   3. Lyse the cell pellet by adding 200 µl of polysome lysis buffer (see Recipes). Mix gently with 200 µl pipette for a number of times and incubate on ice for 15 min.
      
   4. Centrifuge at 10,000 x g for 20 min at 4 °C.
      
   5. Transfer the supernatant to a pre-chilled 1.5 ml tube.
      
   6. Measure OD at 254 nm using a spectrophotometer.
      
   7. Overlay 25-50 OD equivalent of lysate gently on top of the gradient in the ultracentrifuge tube without disturbing the gradient. Leave around 2 mm of space in the gradient tube on top of the lysate layer (Figure 1).
      
   8. Weigh each gradient tube in a balance and adjust the weight of each tube containing the gradient to be exactly equal by adding polysome lysis buffer to the overlaid layer without disturbing the gradient.
      
      
      Figure 1. 10%-50% sucrose gradient showing 2 mm space on top of gradient
      
      
3. Ultracentrifugation and fraction collection
   
   1. Pre-cool the rotor assembly and the tube buckets at 4 °C.
      
   2. Load the tubes carefully into the buckets without disturbing the gradients. Load the buckets on the rotor assembly ensuring equal weights in opposing positions. Perform the ultracentrifugation at 111,000 x g (Avg RCF) for 4 h at 4 °C at maximum vacuum.
      
   3. During the last hour of the ultracentrifugation switch on the gradient profiler, UV detector, gradient profiling software and wash the fractionator tubing with running DEPC treated water for several times and dry by running air.
      
   4. Carefully take out the tubes, one at a time, from the buckets and load into the tube holder of the gradient fractionator, and place at the proper position under the collector piston.
      
   5. Run water through the tubing and press auto zero on the UV monitor.
      
   6. Set up the tube length, gradient length (75 cm for SETON open top polyclear tube), fraction number (12 to 16) in the set up window.
      
   7. Run the fractionation and collect fractions (Figure 2) in 1.5 ml tubes and transfer to ice immediately.
      
      
      Figure 2. Ribosomal fractions from MCF7 cells were analysed by 10-50% sucrose density gradient fractionation. A. Ribosomal RNA content, measured at 254 nm, is plotted against fraction numbers (top panel). B. RNA isolated from selected fractions was analyzed by semiquantitative RT-PCR using PDCD4 and GAPDH primers (bottom panel).
      
      
4. RNA isolation and RT-PCR
   
   1. Add 300 µl phenol (citrate saturated, pH 4.5) and 300 µl chloroform to the tubes containing the gradient fractions.
      
   2. Vortex for 30-60 sec.
      
   3. Centrifuge at 16,000 x g for 15 min at 4 °C.
      
   4. Carefully transfer the supernatants to new tubes.
      
   5. Add 10 µg glycogen (RNase free) and equal volume of isopropanol and mix by inverting the tubes several times. Incubate at room temperature for 30 min.
      
   6. Centrifuge at 16,000 x g for 30 min at 4 °C.
      
   7. Wash the RNA pellet with 1 ml ice cold 75% ethanol.
      
   8. Air dry the RNA pellet and dissolve into 10-20 µl of nuclease free water.
      
   9. Measure the absorbance at 260 nm in a spectrophotometer to determine RNA concentration.
      
   10. cDNA synthesis: Setup the following reaction for cDNA synthesis for each RNA.
       

       RNA	8 µl
       Oligo dT primer	1 µl
       10 mM dNTP mix	1 µl

       65 °C for 5 min. Keep on ice for 1 min.
       Add,
       

       5x RT buffer	4 µl
       100 mM DTT	2 µl
       RNase inhibitor	0.5 µl
       M-MLV RT (200 U/µl)	1 µl
       Nuclease free water	2.5 µl

       Incubate at 37 °C for 1 h. Then at 70 °C for 15 min.
       Store at 4 °C.
       
   11. PCR
       

       10x PCR buffer (with 15 mM MgCl2)	2 µl
       10 µM forward primer for gene of interest/control gene	1 µl
       10 µM reverse primer for gene of interest/control gene	1 µl
       Taq DNA polymerase (1 U/µl)	1 µl
       cDNA	2 µl
       Nuclease free water	13 µl

       Run thermal cycling for 30-35 cycles (Denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 °C for 30 sec) and run the products on agarose gel (Figure 2).

Recipes

1. 10% sucrose and 50% sucrose solution (for 8 ml) (Table 1)
   
   Table 1. Recipe for sucrose density gradient preparation

   	10% sucrose solution (for 8 ml)	50% sucrose solution (for 8 ml)
   Sucrose	0.8 g	4.0 g
   2 M KCl	400 µl	400 µl
   1 M MgCl2	40 µl	40 µl
   100 mM DTT	160 µl	160 µl
   1 M HEPES pH 7.4	160 µl	160 µl
   100 mg/ml Cycloheximide	8 µl	8 µl
   DEPC water	Upto 8 ml	Upto 8 ml

   
   
2. Polysome lysis buffer
   100 mM KCl
   5 mM MgCl₂
   20 mM HEPES (pH 7.4)
   0.5% NP-40
   100 μg/ml CHX
   2 mM DTT
   40 U/ml RNase inhibitor
   1x protease inhibitor cocktail
   

Acknowledgments

We thank members of our laboratory for trying out and standardizing this protocol. Research which led to the development of these protocols was funded by a Wellcome Trust-DBT India Alliance Intermediate fellowship (WT500139/Z/09/Z) to PSR and a CSIR, India Senior Research Fellowship to DKP. This protocol is modified from the protocol described in Merrick and Hensold (2001).

References

1. Merrick, W. C. and Hensold, J. O. (2001). Analysis of eukaryotic translation in purified and semipurified systems. Curr Protocols Cell Biolo 11-9.
   
2. Poria, D. K., Guha, A., Nandi, I. and Ray, P. S. (2016). RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4. Oncogene 35(13): 1703-1715.
   
3. Ray, P. S., Jia, J., Yao, P., Majumder, M., Hatzoglou, M. and Fox, P. L. (2009). A stress-responsive RNA switch regulates VEGFA expression. Nature 457(7231): 915-919.