Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

All animal use was supervised by the Animal Care Committee at the University of Calgary. CD1 mice used for the experiment were purchased from commercial vendor.

1. Preparation of solutions

NOTE To prevent RNA degradation, spray workbench and all equipment with RNase decontamination solution. RNase-free tips are used for the experiment. All solutions are prepared in RNase-free water.

1. Prepare cycloheximide stock solution (100 mg/mL) in DMSO and store at -20 °C.
2. Prepare 2.2 M sucrose stock solution by adding 75.3 g of sucrose to RNase-free water and topping up the volume to 100 mL (for ~16 gradient preparation). The solution can be kept at -20 °C for long-term storage.
3. Prepare 10x salt solution (1 M NaCl; 200 mM Tris-HCl, pH 7.5; 50 mM MgCl₂).
4. Prepare 60% (w/v) sucrose chase solution, containing 30 g of sucrose, 5 mL of 10x salt solution and top the volume up to 50 mL with RNase-free water.
5. Optionally, add a speck of bromophenol blue or approximately 5 µL of 1% bromophenol blue in RNase-free water to the chase solution. 1.6. Store solutions at 4 °C.

2. Preparation of sucrose gradient

NOTE: Accuracy in preparation of sucrose gradients is critical in obtaining consistent and reproducible results.

1. To prepare six 10-50% sucrose gradients, dilute 2.2 M sucrose solution as in Table 1.

Table 1: Sucrose dilutions for preparations of sucrose gradients.

NOTE: Always prepare sucrose gradients in multiples of two to balance weight during ultracentrifugation.

2. Wipe the metal blunt end needle with RNase decontamination solution. Briefly rinse the ultracentrifuge tubes, tubing and the syringe using RNase-free water. Air-dry the tubes such that no water drop remains in the tubes as any remaining water will alter the sucrose concentration.
3. Place the metal needle on the clamp holder and the centrifuge tube on the motorized stage. To prepare gradients consistently, a home-assembled system was used that contains a motorized stage to hold the ultracentrifuge tube and a syringe pump to inject sucrose solutions (Figure 1, see step 9 for details).
4. Fill a 30 mL syringe with ~16 mL of 10% sucrose (enough for six gradients), place it on the syringe pump and connect it to the needle. Make sure that there are no air bubbles in the syringe, tubing, and needle. Wipe the tip off the needle to remove any residual solution.
5. Move the motorized stage up such that the tip of the needle touches the centre of the tube at the bottom.
6. Set the syringe pump at a flow rate of 2 mL/min for a volume of 2.3 mL.
7. After dispensing 2.3 mL of 10% sucrose solution, move down the motorized stage and repeat the process for all six gradients.
8. Repeat steps 2.4-2.7 to add 20% sucrose solution to the bottom of the tubes, followed by 30%, 40% and 50% sucrose solutions similarly.
9. After preparation of the gradient, seal the ultracentrifuge tube using a paraffin film.
10. Leave the tubes overnight at 4 °C such that the different sucrose layers diffuse together to give a continuous gradient.

3. Tissue dissection

NOTE: Pregnant mice were euthanized by cervical dislocation preceded by anesthesia with 5% isoflurane.

1. Collect CD1 mouse embryos at embryonic day 12, or other timepoints as needed, and place embryos in a Ø 10 cm plate containing ice-cold Hank's Balanced Salt Solution (HBSS) on ice to retain cell viability.
2. Under the dissection scope, transfer one embryo to a Ø 6 cm plate containing ice-cold HBSS.
3. Use 21-23 G needles to fix the position of the head by penetrating through eyes at an approximately 45° angle and apply force to make sure the needles are fixed on the plate (Figure 2A).
4. Use No.5 forceps to remove skin and skull, from the middle to the sides.
5. Use forceps to cut the olfactory bulbs and remove the meninges to expose the cortical tissues.3.6.Use curved forceps to cut the cortical tissues into 2-3 mL of neurobasal medium on ice (Figure 2B). Pool cortical tissues from different embryos as needed. Tissues from 8-10 embryos usually give ~200 µg total RNA.

4. Cell lysis

1. On the day of tissue dissection, prepare fresh cell lysis buffer as described in Table 2.

Table 2: Preparation of polysome lysis buffer.

NOTE: Supplement lysis buffer with protease and phosphatase inhibitors.

2. Add cycloheximide to the neurobasal medium with dissected tissues to a final concentration of 100 µg/mL and incubate at 37 °C for 10 min. Cycloheximide blocks translation elongation and, therefore, prevents ribosome run-off ¹⁵.
3. Centrifuge at 500 x g for 5 min at 4 °C and discard the supernatant.
4. Wash the tissues twice with ice-cold phosphate-buffered saline (PBS) supplemented with 100 µg/mL cycloheximide.
5. Add 500 µL of cell lysis buffer supplemented with 4 µL of RNase inhibitor. Pipette up and down to resuspend the tissue in lysis buffer. Use an insulin needle to gently lyse the tissue.
6. Incubate the tissues on ice for 10 min with brief vortexing every 2-3 min.
   NOTE: To prevent tissue degradation, ensure that all steps of tissue lysis are carried out on ice.
7. Centrifuge at 2,000 x g for 5 min at 4 °C.
8. Transfer the supernatant to a new centrifuge tube on ice.
9. Centrifuge at ~13,000 x g for 5 min at 4 °C.
10. Transfer the supernatant to a new centrifuge tube on ice.
11. Measure the RNA concentration in the tissue lysate using a UV-Vis spectrophotometer.
    NOTE: If multiple samples are included in the experiment, dilute samples to the same concentration with extra cell lysis buffer to minimize variation.

5. Sample loading and ultracentrifugation

1. Pre-cool the ultracentrifugation rotor and swing buckets at 4 °C, and set the temperature of the ultracentrifuge to 4 °C.
2. Keep 20 µL of the tissue lysate as total RNA input.
3. Load samples (50-300 µg RNA with an equal volume) on the top of the sucrose gradients by slowly dispensing the lysate to the walls of the ultracentrifuge tubes.
4. Gently place the ultracentrifuge tubes in the swing bucket. Ensure that all diametrically opposite buckets are balanced.
5. Load the swing buckets on the rotor. Set the ultracentrifuge to 190,000 x g (~39,000 rpm) at 4 °C for 90 min.
6. Gently place the gradients on ice after centrifugation.

6. Fractionation and sample collection

NOTE: A home-assembled fractionating, recording and collecting system is used for the analysis and collecting samples from the gradients (Figure 3, see Device components).

1. Place an empty ultracentrifuge tube on the tube piercer and gently penetrate the tube with the needle from the bottom.
2. Switch on the UV monitor, and open the digital signal recording software.
3. Fill a 30 mL syringe with 25 mL of 60% sucrose chase solution. Gently press the syringe such that the chase solution fills up the empty tube and go through the 254 nm UV monitor to set the baseline for detection.
4. Press auto-zero on the UV-monitor to register a baseline for detection. Press play on the software to begin recording.
5. Once the system establishes a baseline, pause recording on the software and retract the chase solution. Ensure that no residual chase solution remains in the system.
   NOTE: Rinse the system with RNase-free water to remove residual sucrose solutions remaining from the previous run.
6. Load one sample tube to the tube piercer. Gently penetrate the tube with the needle from the bottom.
7. Begin recording on the software. Start the syringe pump (flow rate of 1 mL/min for a volume of 25 mL) and the fraction collector to collect polysome fractions. Set fractionator settings to 30 s.
   NOTE: Before each run, ensure no air bubbles in the syringe or the tubing by gently pressing the syringe to let the chase solution flow continuously through the needle.
8. Collect the fractions into 1.5 mL tubes (500 µL each) using a fraction collector.
9. The fractions can be processed immediately or stored at -80 °C.
10. After fraction collection, rinse the system with RNase-free water to remove any remnant sucrose.

7. Extraction of RNA

1. To each fraction, add 10 ng of luciferase mRNA spike-in control.
2. Add three volumes of guanidium hydrocholride based commercial RNA isolation reagent to each fraction.
3. Vortex briefly for 15 s.
4. Extract RNA using an RNA extraction kit compatible with the solution used in 7.2.

8. Reverse transcription and real-time PCR

1. Measure the concentration of the RNA using UV-Vis spectrophotometer.
2. Subject RNA to reverse transcription using a cDNA synthesis kit, according to manufacturer's protocol.
3. Use quantitative real-time PCR (qPCR) to examine the polysomal distribution of gapdh mRNA as an example. qPCR was performed using a qPCR detection system and a qPCR mastermix reagent, with the following cycling conditions: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 40 s, followed by 95 °C for 60 s.
4. Obtain Threshold cycle (Ct) values from the amplification plots and used to calculate fold change using ΔΔCt method.

9. Sucrose gradient making system assembly

NOTE: Follow the steps to assemble each component (as described in Table 3) of the sucrose gradient maker (Figure 1).

1. Mount two vertical brackets (A2) on the base breadboard (A1).
2. Use two slim right-angle brackets (B2) to mount the linear stage actuator to the base breadboard (A1).
3. Use the setscrew on the Ø12.7 mm aluminum post (C2) to fix it on the tube holder base breadboard (C1) as a stand for the tube holder.
4. Assemble the right-angle Ø1/2" to Ø6 mm post clamp (C3) with the post (C2) and the mini-series optical post (C4).
5. Use the setscrew on the optical post (C4) to connect a small V-clamp (C5) as the tube holder.
6. Put a centrifuge tube in the tube holder and adjust the angle to make it vertical. Mark the position of the tube and mount the pedestal post holder (C6) on the base breadboard (C1) to support the tube.
7. On the other side of the breadboard (A1), use the setscrew of the Ø12.7 mm aluminum post (D1) to fix it and connect a mini-series optical post (D3) using a right-angle Ø1/2" to Ø6 mm post clamp (D2).
8. Connect the miniature V-clamp (D4) with blunt-end needle (D5) to the post (D3). Adjust the angle of the clamp to set the needle vertical, and adjust the length of post to ensure the needle meets the centrifuge tube.
9. Connect the actuator (B1) to the stepper motor driver (E1) and use the UNO R3 controller board and the joystick module from the UNO starter kit (E2) to control the actuator. A power adaptor (e.g., 9-24 V AC/DC adjustable power adaptor) can be used to drive the motor separately.
10. Place the syringe pump (F) next to the gradient making station and connect the syringe with the needle.
11. Control the tube holder stage up and down using the joystick.

Table 3: Gradient making system components.

10. Fractionating and detecting system assembly (Figure 2).

1. Use a regular round jaw burette clamp to mount the optics module of the UV monitor on the tube piercer. Attach one end of the tubing (1 cm long and 0.56 mm internal diameter) to the connecter of the tube piercer and the other end to the Fluid In port of the optics module. Connect the Fluid Out port to the fractionator.
2. Connect the optics module with the control unit of the UV monitor according the manual of the manufacturer.
3. Use a breakout cable to connect the signal output socket to the digital converter at the ground and analog input connections, according to the manufacturer's manual.
4. Connect the digital converter to a laptop using a regular USB cable, and record the converted digital signals using data acquisition software.