NIH, NIA-IRP PROTOCOL
Laboratory of Genetics
RNA Regulation Section Polysome Fractionation
Gradient Preparation: Day BEFORE fractionation
• Prepare sucrose gradients:
o Using prepared solutions containing 10%, 20%, 30%, 40% or 50% sucrose (see chart below),
begin by adding 2.2 ml of 10% sucrose into each tube (Beckman # 331372).
o Underlay each subsequent layer (2.2 ml of 20%, 30%, 40%, then 50%) using a 9” pasteur pipet
and manual pipettor, being careful not to introduce any air bubbles.
o Leave gradients at 4oC overnight to allow the gradient to become linear.
o The gradients are OK to use within 16 hours - 3 days (at 4oC) or may be frozen at -20oC or -80oC
for 2 weeks (or more) and thawed right before use.
• Pre-cool the SW41Ti rotor and buckets at 4oC overnight.
Lysate preparation:
1. Add 100 g/ml cycloheximide (CHX) directly to the cell culture media. Incubate 10 min at 37 oC.
2. Remove media. Wash cells with ice cold PBS + 100 g/ml CHX.
3. Trypsinize cells*. Transfer the cells to 15 ml tube using cold PBS + CHX to rinse the plate.
• *NOTE: for differentiated cells (neurons, muscle) or senescent cells, it is better to add PEB
directly to the plate, scrape the cells, and transfer to a microfuge tube. Continue from step 6.
4. Spin briefly (1200 rpm, 5 min). Remove the media and place the pellet on ice.
• Cell pellets can be frozen at -80oC, stored or shipped.
5. Resuspend the pellet in 0.5 – 1.0 ml PEB (+ protease and RNase inhibitors + CHX). Transfer to a
microfuge tube.
6. Incubate on ice for 10 min with occasional inverting.
7. Spin 10,000 rpm for 10 min at 4oC to pellet nuclei and debris.
8. Transfer the supernatant (cytoplasmic lysate) to a fresh tube.
9. Measure the protein concentration by Bradford assay and/or the total RNA concentration by Nanodrop.
This supernatant can also be frozen at -80oC and fractionated another time (but fresh is best).
Fractionation:
• Carefully layer equal amounts of cytoplasmic lysate (1 ml or less containing 1.0 – 1.5 mg protein or 100-
300 g RNA) to the top of a 10-50% sucrose gradient. Be sure all tubes are equally balanced.
• Using a Beckman ultracentrifuge, centrifuge the gradients for 90 min in a SW41Ti swinging bucket rotor
at 39,000 rpm, 4oC, max accel & brake.
• Keep the gradients on ice after centrifuging. To begin, place the gradient onto the fractionating system
- the piercing needle will puncture the bottom of the tube.
• Collect 1 ml/min fractions into tubes (12 fractions total per gradient). Transfer fractions to an ice
bucket. Analyze the fractions as desired or freeze the fractions until ready to use.
Polysome Fractionation page 2
Fraction Analysis:
Protein analysis:
Run equal amounts of each fraction on SDS-PAGE gels to analyze by western blotting.
The fractions may require TCA precipitation prior to western analysis.
RNA preparation: Use equal volumes of each fraction for all steps.
• Use 0.5 ml of each sucrose fraction
• Add 0.5 ml Trizol (Thermo Fisher Scientific # 15596018)
• Add 200 l chloroform
• Vortex or shake vigorously
• Spin at max speed for 15 min at 4oC
• Collect 500 l of supernatant – transfer to a fresh microfuge tube. Discard lower (red) layer.
• Add 1 ml isopropanol plus 2 l Glycoblue (Ambion)
• Incubate at -20oC overnight.
• Spin at max speed for 15 min at 4oC
• Wash pellet once with 1 ml ice-cold 70 % ethanol
• Spin at max speed for 5 min at 4oC
• Remove ethanol. Do a short spin at 4oC, remove remaining ethanol
• Air dry RNA pellet for 5-10 min at RT
• Dissolve RNA in 11 l H2O
• Use RNA for reverse transcription followed by real-time quantitative (q)PCR analysis, for RNA gel
electrophoresis, or for northern blotting.
• Do NOT measure RNA concentrations as this will vary between fractions, with some fractions having
very little RNA.
RT-qPCR analysis:
• Prepare cDNA for each fraction using 10 l RNA for each fraction (as prepared above).
• Dilute the cDNAs (20 l volume) with RNase-free water to 100 l final volume
• Using the cDNAs from each fraction, prepare qPCR reactions to analyze specific mRNAs of interest
as well as 1-2 housekeeping genes (Actin and/or Gapdh).
• Using the Ct values, calculate the % distribution for the mRNAs across the gradients.
• There is no normalization involved, just confirm that the housekeeping genes are similarly
distributed across the gradients for different treatment conditions, then any observed changes
in distribution curves seen for other mRNAs of interest with the different treatments can be
deemed to be specific.
• Sample calculations are shown on page 5
Revised May 2016
Polysome Fractionation page 3
Reagents and Buffers
2.2 M Sucrose (MW 342.3) - store at 4oC. Prepare a suitable volume:
753.06 g / L = 564.795 g / 750 ml = 376.54 g / 500 ml
10X Salts Solution (gradient additive) - store at 4oC
50 ml Final 10X concentration 1X
5 M NaCl 10 ml 1M 100 mM
1 M Tris-HCl pH 7.5 10 ml 200 mM 20 mM
1 M MgCl2 2.5 ml 50 mM 5 mM
H2O 27.5 ml
Chase Solution (60% sucrose): 40 ml 2.2M sucrose
5 ml H2O
5 ml 10X Salts Solution
50 ml total volume + speck of BPB => Store at 4oC
Polysome Extraction Buffer (PEB) (cytoplasmic lysate)
500 ml (store at 4oC)
20 mM Tris-HCl (pH 7.5) 10 ml 1M
100 mM KCl 50 ml 1M
5 mM MgCl2 2.5 ml 1M
0.3 % Igepal CA-630 1.5 ml stock
H2O to 500 ml
• ADD 25X protease inhibitors, RNase inhibitor, and 100 g/ml CHX to an aliquot of PEB before use
Cycloheximide (CHX) (1000X)
Stock solution is 100 mg/ml in DMSO
Aliquot and store at -20oC
Complete Protease Inhibitor Cocktail (25X)
Sigma / Roche #4693132001
(25X = 1 tablet per 2 ml)
Beckman SW 41Ti swinging bucket rotor with buckets
Centrifuge tubes # 331372 (14x 89 mm polyallomer)
RiboLOCK RNase Inhibitor
Thermo Fisher Scientific # EO0384 (24 x 2500U)
GlycoBlue
Thermo Fisher Scientific # AM9516 (5 tubes)
Polysome Fractionation page 4
Sucrose Gradients
For 18 Gradients:
Solution 10% 20% 30% 40% 50%
2.2M Sucrose 6 ml 12 ml 18 ml 24 ml 30 ml
H2O 34.5 ml 28.5 ml 22.5 ml 16.5 ml 10.5 ml
10X salts solution 4.5 ml 4.5 ml 4.5 ml 4.5 ml 4.5 ml
TOTAL volume 45 ml 45 ml 45 ml 45 ml 45 ml
For 72 Gradients:
Solution 10% 20% 30% 40% 50%
2.2M Sucrose 24 ml 48 ml 72 ml 96 ml 120 ml
H2O 138 ml 114 ml 90 ml 66 ml 42 ml
10X salts solution 18 ml 18 ml 18 ml 18 ml 18 ml
TOTAL volume 180 ml 180 ml 180 ml 180 ml 180 ml
Polysome Fractionation page 5
Sample calculations for the % distribution of a specific mRNA across the gradient:
delta Ct %mRNA
Avg CT(frac X) - Avg Ct(fract 1) [2^(-deltaCt) fraction X]*100/sum
Fraction RNA AVG Ct delta Ct 2^(-deltaCt) %RNA
1 RNA2 26.537 0.000 1.0 0.00
2 RNA2 15.731 -10.806 1790.5 0.12
3 RNA2 14.958 -11.579 3059.3 0.21
4 RNA2 11.411 -15.125 35744.5 2.49
5 RNA2 9.501 -17.036 134371.4 9.37
6 RNA2 11.969 -14.568 24288.7 1.69
7 RNA2 7.293 -19.244 620929.5 43.31
8 RNA2 10.353 -16.184 74461.8 5.19
9 RNA2 7.620 -18.917 494971.8 34.52
10 RNA2 11.364 -15.173 36941.8 2.58
11 RNA2 14.363 -12.174 4619.9 0.32
12 RNA2 15.252 -11.285 2494.5 0.17
1433674.8 100.00
sum of all 2^(-dCt)
Graph: % mRNA as a scatter plot with curved lines
50.00
45.00
% RNA2 in each fraction
40.00
35.00
30.00
% mRNA
25.00
20.00
15.00
10.00
5.00
0.00
-5.00 0 2 4 6 8 10 12
Fraction
