Translational regulation plays an important role in brain development. Polysome profiling provides a useful and powerful tool to assess the translational status of mRNA, making it possible to examine translational control functions during brain development. This method is an easy and low-cost approach to make sucrose gradients and assess polysome fractionation.
In this protocol, it is used to analyze the developing mouse cortex. However, it can be easily adapted to study other systems. Begin by diluting 2.2 molar sucrose to prepare six 10 to 50%sucrose gradients.
Fill a 30 milliliter syringe with approximately 16 milliliters of 10%sucrose, place it on the syringe pump and connect it to the needle. Make sure that there are no air bubbles in the syringe, tubing or needle, and wipe the tip of the needle to remove any residual solution. Move the motorized stage up such that the tip of the needle touches the center of the tube at the bottom.
Set the syringe pump to a flow rate of two milliliters per minute for a volume of 2.3 milliliters. After dispensing 2.3 milliliters of 10%sucrose solution, move down the motorized stage and repeat the process for all six gradients. Then add 20%sucrose solution to the bottom of the tubes followed by 30, 40 and 50%Collect CD1 mouse embryos at embryonic day 12 and place them in a 10 centimeter plate containing ice cold HBSS on ice.
Under the dissection scope, transfer one embryo to a six centimeter plate containing ice cold HBSS. Use 21 to 23 gauge needles to fix the position of the head by penetrating through the eyes at a 45 degree angle and apply a force to make sure the needles are fixed on the plate. Use number five forceps to remove the skin and skull working from the middle to the sides, then cut the olfactory bulbs and remove the meninges to expose the cortical tissues.
Use curved forceps to cut the cortical tissues into two to three milliliters of neurobasal medium on ice. Pull the tissues from different embryos as needed. Tissues from eight to 10 embryos usually give approximately 200 micrograms of total RNA.
Add cycloheximide to the neurobasal medium with the dissected tissues to a final concentration of 100 micrograms per milliliter and incubate at 37 degrees Celsius for 10 minutes. Centrifuge the tissue at 500 x g for five minutes at four degrees Celsius and discard the supernatant. Wash the tissues twice with ice cold PBS supplemented with 100 micrograms per milliliter cycloheximide.
Add 500 microliters of cell lysis buffer supplemented with four microliters of RNase inhibitor and pipette up and down to resuspend the tissue. Use an insulin needle to gently lyse the tissue. Then incubate on ice for 10 minutes with brief vortexing every two to three minutes.
After the incubation, centrifuge the lysate at 2000 x g for five minutes at four degrees Celsius and transfer the supernatant to a new centrifuge tube on ice. Repeat this process once more centrifuging at 13, 000 x g. After transferring the supernatant to a new centrifuge tube on ice, measure the RNA concentration in the tissue lysate using a UV-Vis spectrophotometer.
Load the samples on top of the sucrose gradients by slowly dispensing the lysate to the walls of the ultracentrifuge tubes. Gently place the sucrose gradients in the buckets and ultracentrifuge the samples at 190, 000 x g and four degrees Celsius for 90 minutes. Place an empty ultracentrifuge tube on the tube piercer and gently penetrate the tube with the needle from the bottom.
Fill a 30 milliliter syringe with 25 milliliters of 60%sucrose chase solution. Then gently press the syringe such that the chase solution fills the empty tube and goes through the 254 nanometer UV monitor to set the baseline for detection. Press auto zero on the UV monitor to register a baseline for detection, then press Play on the software to begin recording.
Once the system establishes a baseline, pause recording on the software and retract the chase solution, making sure that no residual chase solution remains in the system. Load one sample tube on the tube piercer and gently penetrate the tube with the needle from the bottom. Begin recording on the software, make sure that there are no air bubbles in the line.
Then start the syringe pump and the fraction collector to collect polysome fractions, setting the fractionator to 30 seconds. Collect the fractions into 1.5 milliliter tubes. The cortical lysate containing RNA pulled from eight embryos was separated by a sucrose gradient into 12 fractions.
Peaks of UV absorbance at 254 nanometers identified fractions containing the 40S subunit 60S subunit and 80S monosome and polysomes. Analysis of the fractions by Western blot for the large ribosomal subunit Rpl10 showed its presence in the 60S subunit monosomes and polysomes. In contrast, cytoplasmic proteins, Gapdh and Csde1, were not associated with ribosomes, but were enriched in fractions containing free RNA.
Consistent with the separation of proteins in different fractions, Gapdh and sox2 mRNAs were highly enriched in the fractions containing heavy polysomes, suggesting that these mRNAs are efficiently translated in the developing cortex. In contrast, rpl7 and rpl35 mRNAs were enriched in the fraction containing monosomes, suggesting repressed translation. To obtain reproducible results, it is important to maintain consistency in making sucrose gradients.
The syringe pumps should be used to eject the sucrose. During fractionation and sample collection, it is important to wash the system with RNase-free water. This reduces any residual sucrose remaining from the previous sample.
This method can be used to assess the translational status of mRNAs in various tissues. Using the platform, consistent gradients for polysome profiling can be obtained, which provides a low-cost solution for this classical and useful technique.
