To begin, collect the frozen heat-stressed and untreated five-day-old Columbia-0 Arabidopsis seedlings into the liquid nitrogen container. Grind the seedlings using a homogenizer into powder for one minute. Then add 500 microliters of polysome extraction buffer to the tube.
Invert and vortex the tube to mix the sample. Centrifuge the extraction solution at 12, 000G for 10 minutes at four degrees Celsius. Then, filter the supernatant through a 100-micrometer cell strainer to obtain a clear, particle-free extraction solution.
Load the filtered supernatant onto the pre-prepared sucrose gradient and wait until it reaches equilibrium. Then, ultracentrifuge the gradient using a swinging bucket rotor at 210, 000G for 3.5 hours at four degrees Celsius with maximum acceleration and deceleration rates. The non-polysomal and polysomal RNA are separated based on their density in the corresponding sucrose gradient solution.
Prepare the chase solution for the micro-volume syringe pump consisting of 70%sucrose, 10%glycerol, and 0.02%bromophenol blue. Mix the solution thoroughly for 30 to 40 minutes. After injecting the chasing solution into the gradient, using the software, control the density gradient fractionator.
Then calibrate the machine with deionized water. After ultracentrifugation, place the tube onto the density gradient fractionator. Using the tube-piercing system, connect the tube to the syringe pump and the ultraviolet detector.
Switch the density gradient fractionator to software control mode remote. Set the injection flow rate of the micro-volume syringe pump to three milliliters per minute. Initiate the process to obtain the polysome profile graph by measuring the optical density at 254 nanometers using the fractionator.
Based on the polysome profile measurements, collect the non-polysomal and polysomal RNA fractions separately. Thoroughly mix the collected RNA fractions with pre-cold two-times high-salt solution. Then add eight microliters of a diluted stock of eukaryotic poly-A RNA control from the kit.
Centrifuge the high-salt solution mixed RNA with a swinging bucket rotor at 450, 000G for five hours at four degrees Celsius. Carefully pour off the supernatant and wash the RNA pellet three times with 500 microliters of pre-cold DEPC-treated water. After the last wash, add 500 microliters of RNA extraction reagent to the RNA sample.
Mix and incubate for 10 minutes. Add 100 microliters of chloroform and mix thoroughly. Incubate for 10 minutes to allow phase separation.
Centrifuge the mixture at 12, 000G for 10 minutes at four degrees Celsius. Transfer the upper clear aqueous layer containing RNA into a new tube and mix it gently with an equal volume of isopropanol. Incubate the mixture at minus 20 degrees Celsius for two hours to precipitate the RNA.
After precipitation, centrifuge again and discard the supernatant, leaving the RNA pellet at the bottom. Wash the RNA pellet with one milliliter of pre-cold 75%ethanol. Centrifuge at 12, 000G for 10 minutes at four degrees Celsius and air-dry the RNA pellet.
Upon drying, resuspend the pellet in 30 microliters of DEPC-treated water. Measure the RNA concentration using a full-spectrum spectrophotometer at 220 to 750 nanometers, focusing on optical density at 260. The polysome profiling showed a distinct separation between the non-polysomal and polysomal fractions under normal conditions.
Heat stress at 40 degrees Celsius significantly reduced the signal intensity of the polysomal fraction, and this effect was reversed after a two hour recovery at 22 degrees Celsius, bringing the signal back to control levels. RNA extraction results indicated that heat stress reduced the percentage of RNA in the polysomal fraction, which was restored after recovery at 22 degrees Celsius.
