The following procedure is for a monolayer cell culture grown in a 15-cm tissue culture plate. This protocol can also be adapted to cells transfected using a calcium phosphate protocol (Graham and van der Eb, 1973, Jin et al., 2003, Bor et al., 2006).
Growing cells TIMING 1-3 d
1| Seed 0.8-1.0 X 107 293T cells onto each 15-cm culture dish 1-3 days before polysome fractionation. At harvest, the cells should be at no more than 70-80% confluency.
CRITICAL STEP: When cultures approach 100 % confluency, they tend to slow down general protein translation, and polysomes extracted from such cells show a shift from actively translating polysomes to a predominance of monosomes (a large 80S peak in the polysome profile). It is therefore important that the number of cells seeded are adjusted as necessary to avoid them reaching stationary phase.
Polyribosome fractionation TIMING 5-6 h
2| 48 h post seeding, add cycloheximide (100 mg/ml) to 50 µg/ml final concentration to the growth medium. Incubate at 37oC for 30 min.
CRITICAL STEP: Cycloheximide is an inhibitor of protein biosynthesis and exerts its effect by interfering with peptidyl transferase on the 60S ribosome, thus blocking translational elongation. Addition of cycloheximide “freezes” the ribosomes on the mRNA, preventing them from completing translation and falling off the RNA during the polysome extraction procedure. However, prolonged exposure to cycloheximide is toxic to the cells and should be avoided.
3| Transfer plate to rest directly on ice in a box or bucket. Aspirate growth medium, and wash cells with 10 ml of ice-cold PBS containing 50 µg/ml of cycloheximide. Aspirate the solution, and repeat the wash step.
CRITICAL STEP: Keep cells and solutions on ice as cold temperature will slow down translation elongation and RNase activity.
4| Scrape cells with a cell lifter, and transfer to a 1.5 ml microcentrifuge tube. Centrifuge in a refrigerated microcentrifuge at 7000 rpm for 1 min. Aspirate supernatant.
5| Add 250 µl of 2X RSB/RNasin to resuspend cell pellet. Add 250 µl of polyribosome extraction, and mix quickly.
6| Incubate on ice for 10 min. Spin for 10 sec to pellet nuclei. Transfer 500 µl of cytoplasmic extract to a new microcentrifuge tube. Centrifuge at 10,000 X g for 10 min at 4oC.
7| While the cytoplasmic extract is centrifuging, prepare gradients in an ultracentrifuge tube by underlaying 5.5 ml of 10% sucrose solution with 5.5 ml of 50% sucrose solution without disturbing the interface. This is done by gently inserting the tip of a blunt end stainless steel needle to the bottom of the tube and slowly releasing the 50% sucrose solution from the syringe to displace the 10% sucrose solution upwards in the tube.
8| Close the tube with a BioComp cap, and put the capped tubes in the BioComp gradient master (Fig 1a). Select the gradient parameter according to the manufacturer’s instructions, and start the run. When the gradient formation is finished, carefully remove the cap, and transfer the tube to an SW41Ti rotor bucket. Carefully layer on top of gradient an additional 500 µl of 10% sucrose.
CRITICAL STEP: During cap insertion, make sure there are no air bubbles trapped inside the tube, since free-floating bubbles have a deleterious effect on gradient formation. Keep rotor buckets cold throughout the procedure.
9| When the spin is finished, carefully layer 500 µl of the cytoplasmic extract (prepared as described in step 6) on top of the sucrose gradient.
(Optional) To disrupt polyribosomes in a separate control experiment, add 30 µl of 250 mM EDTA to 470 µl of cleared cytoplasmic extract (15 mM final concentration), and layer the extract on top of a second gradient (Calzone et al., 1998; Johannes and Sarnow, 1998). This concentration of EDTA does not generally disrupt mRNP complexes.
10| Place the rotor buckets in the SW41Ti rotor, and centrifuge at 36,000 rpm for 2 h at 4oC. When the centrifugation is finished, take off rotor buckets, and keep them in ice until fractions from each tube are collected (to minimize potential RNAse activity) .
11| Collect approximately 20 550 µl fractions on ice into 1.5-ml microcentrifuge tubes using the BioComp piston gradient fractionator (Fig. 1b) equipped with a UV-M II monitor connected to the DATAQ DI-154RS data acquisition module. This allows the concomitant measurement of absorbance at 254 nm and converts the analog signal from the UV monitor into a digital format so that it can be viewed and recorded on a PC using WinDaq/100 data acquisition software. (see Figure 3 for an example of the UV absorbance profile). Further information about this setup is available from BioComp or the authors.
12| Add 60 µl of 10% SDS and 12 µl of Proteinase K (10 mg/ml) to each fraction, mix and incubate for 30 min at 42oC.
PAUSE POINT: Samples can be stored at –80oC.
RNA isolation and precipitation TIMING 2-8 h, depending on number of gradients
13| Transfer 300 µl of each Proteinase K-treated fraction to a new microcentrifuge tube, and extract each fraction twice with 300 µl of phenol/chloroform/isoamyl alcohol and once with 300 µl of chloroform/isoamyl alcohol. Keep tubes on ice.
CAUTION: Phenol is readily absorbed through the skin and can cause severe burns to the eyes and skin. Chloroform is a skin and eye irritant, and it is a suspected human carcinogen and reproductive hazard. Adding chloroform to phenol enhances the ability of phenol to be absorbed by the skin. Wear gloves at all times. Tips and tubes that have contacted these organic solvents should be disposed of appropriately.
14| Transfer each supernatant to a new microcentrifuge tube. Precipitate RNA from each fraction by the addition of 30 µl of 3 M sodium acetate (pH 5.2) and 825 µl of 100% ethanol. Mix and store the solution at –80oC for 15 min.
PAUSE POINT: Fractions can be stored at –80oC.
15| Collect RNA by centrifuging at 13000 rpm for 20 min at 4oC. Wash the RNA pellets with1 ml of 75% ethanol. Carefully aspirate supernatant from each tube, leaving the cap open to allow the RNA pellet to dry.
CRITICAL STEP: Do not overdry the RNA pellet, because doing so will make the pellet difficult to resuspend.
(Optional) DNase I treatment and RT-PCR TIMING 6-8 h
For RNA to be analyzed by RT-PCR, resuspend the RNA in 20 µl of 1X DNase1 buffer and 1 unit of DNase I, and incubate at 37oC for 1 hour. At the end of incubation, add 180 µl of DEPC water to the sample, and extract with 200 µl of phenol/chloroform/isoamyl alcohol twice and 200 µl of chloroform/isoamyl alcohol once. Precipitate RNA by adding 20 µl of 3M sodium acetate (pH 5.2) and 550 µl of 100% ethanol, and store the solution at –80oC for 15 min. Collect RNA by Centrifuging at 13000 rpm for 20 min at 4oC. Wash the RNA pellet with 1 ml of 75% ethanol. Carefully aspirate supernatant from tube. Dry the RNA pellet. Resuspend the RNA pellet with 20 µl of DEPC-water.
RT-PCR set up
RNA/ 1.0 µl
DEPC-water/ 11. 55 µl
10X Taq DNA polymerase buffer/ 2.0 µl
50 mM MgCl2/ 0.8 µl
10 mM dNTP solution/ 0.4 µl
0.2 M DTT /2.0 ml
Primer 1 (100 ng/ml)/ 1.0 µl
Primer 2 (100 ng/ml)/ 1.0 µl
RNasin (40 U/µl) 0.05 µl
Taq DNA polymerase (5 U/µl) 0.1 µl
AMV RT (24 U/µl) 0.1 µl
Prepare a master mix for multiple reactions to minimize reagent loss and enable accurate pipeting.
Incubate tubes in a themocycler at 48oC for 45 min to synthesize the first-strand cDNA, followed by 95oC for 3 min to inactivate the activity of AMV reverse transcriptase. Perform 15-25 cycles of PCR amplifications as follows:
Denature at 94oC for 30 sec
Anneal at 55oC for 30 sec
Extend at 72oC for 90 sec
At the end of PCR cycles, continue incubation for another 7 min at 72oC. Analyze PCR products by agarose gel electrophoresis, and visualize by ethidium bromide staining.
CAUTION: Ethidium bromide is mutagenic. Wear gloves at all times.
Formaldehyde gel electrophoresis of RNA from polysome gradient fractions TIMING 5-6 h
16| Resuspend the RNA pellet in 4 µl of DEPC-water, and 15.5 µl of RS buffer. Incubate samples at 55oC for 10 min, cool on ice, add 2 µl of 10X RNA loading dye, and load onto a 1% agarose gel containing formaldehyde. Run gel at 120 volts for 3 h with a peristaltic pump that recirculates running buffer between the electrodes.
Preparation for transfer TIMING 3 h
17| Turn off power supply for electrophoresis. Soak the gel in 250 ml of autoclaved water for 15 min, changing the water 3 times. This step is carried out in a tray with gentle rocking.
18| Denature the gel by completely immersing it in 50 mM NaOH for 20 min.
19| Neutralize the gel in 100 mM Tris-HCl (pH 7.0-7.6) for 20 min.
20| Soak the gel in 10X SSC for 15 min.
21| Prepare nylon membrane by cutting to the size of the gel, and soak the membrane in 100 ml of 10X SSC.
Transfer TIMING 16 h
22| Set up capillary transfer in the following order (Fig 2):
Stack at least 3 inches of cut-to-size paper towels in a Pyrex tray.
Soak 3 pieces of Whatman paper in 10X SSC for 3 min, and lay them on top of paper towels.
Place the nylon membrane on top of the Whatman paper.
Lay gel top-side facing upward, and remove bubbles between gel and membrane.
Soak 3 pieces of Whatman paper in 10X SSC for 3 min, and lay them on top of the gel.
Add 20X SSC to two reservoirs, and place one on each side of the Pyrex tray. Soak a long piece of Whatman paper in 20X SSC for use as a wick. Lay the wick on top of the Whatman paper with ends dipping into the reservoirs.
Place a plastic or glass plate on top of the wick. Weigh the whole set-up with 4 plastic bottles, each containing 100 ml of water.
Allow the RNA to transfer overnight.
CRITICAL STEP: Avoid trapping air bubbles between the filter papers and nylon membrane by rolling a clean plastic pipette across. Any bubbles will lead to uneven or poor transfer. Once it is placed, do not move the membrane as some transfer of RNA may have already taken place. Also, be sure not to allow the wick to collapse and touch the towel stack as this will short out the capillary action and also result in a poor transfer.
Post-transfer processing TIMING 30 min
23| At the end of the transfer period, lift the nylon membrane, and rinse it briefly in 10X SSC.
24| Place the nylon membrane on a piece of Whatman paper with the RNA side facing upwards. Cross-link RNA to the nylon membrane using a UV cross-linker at a setting of 120 mjoules/cm2.
Blocking and preparation of radioactive probe TIMING 1-4 h
25| Put the membrane in a heat-sealable bag. Add pre-hybridization solution to the bag, and seal the bag. Incubate the bag at 43oC for 1-4 h in a slowly shaking water bath.
26| Label the DNA fragment with [alpha-32P]dCTP using the RediPrime II kit according to the manufacturer’s instructions. Purify the radiolabeled probe from unincorporated nucleotide using the NICK column according to the manufacturer’s instructions. Use 1 µl of probe to determine the counts with a scintillation counter. Calculate the total incorporation of [alpha-32P]dCTP and the specific radioactivity of the probe.
CRITICAL STEP: For a good probe, the specific radioactivity should be around 1 X 109 cpm/µg of template.
Hybridization TIMING 16 h
27| Replace the heat-sealable bag with hybridization solution. Denature the probe by placing it in a boiling water bath for 5 min followed by snap cooling on ice. Briefly spin down the probe. Add denatured probe to the bag, and seal it. Continue hybridization overnight at 43oC in a water bath with slow agitation.
Post-hybridization washing TIMING 2-3 h
28| Remove the nylon membrane from the bag to a Rubbermaid container, and rinse it briefly with 6X SSC/0.1%SDS twice. Using the same solution, wash the membrane three times, 15 min each, at room temperature. Then wash the membrane once at 43oC for 15 min.
29| Wash the membrane with 0.1X SSC/0.1% SDS at room temperature, for 15 min each, two or three times.
30| Check the background of the membrane using a Geiger Counter. If the background is high, continue the wash with 0.1X SSC/0.1% SDS at 43oC for 15 min.
CRITICAL STEP: The needed stringency of the washes depends on several factors (e.g., the size of the probe and G/C content). If the washes are too stringent, the signal might be too low. If the washes are not stringent enough, the background might be too high.
RNA detection TIMING 1 hour to 1 week, depending on hybrid yield and probe specific activity
31| Remove the membrane, and wrap it with plastic wrap. Place it onto a PhosphorImager cassette, and flatten it against a PhosphorImager screen.
32| Depending on hybrid yield and specific activity of the probe, scan the screen for the appropriate amount of time. Analyze the data with ImageQuant software.