Hi, I'm Cecile. I work at the LGBP in Marseilles. The protocol we are going to describe in this video is a simple method to isolate polysomes and monosomes from various plant tissues in order to identify mRNAs that are actively translated and to study translation regulation.
As an example, we are going to show you the isolation of polysomes from six days'old Arabidopsis thaliana seedlings, subsequent isolation of RNA and the analysis of the results. Sow the seeds on plate and leave them two days at four degrees. Then grow the plants during six days.
Harvest the seedling and grind them in liquid nitrogen. Weight 300 milligrams of plant powder and add 2.4 milliliter of polysome buffer. Centrifuge to collect plant fragments and load the supernatant on the top of a sucrose gradient.
Ultra-centrifuge gradient then collect it from the bottom to the top with continuous reading of the OD.Pull the fraction into a polysome, a monosome and a supernatant fraction and precipitate RNA overnight. Ultra-centrifuge, resuspend the pellet and extract RNA for subsequent analysis. A few days before performing polysome profiling, prepare the sucrose gradient.
Prepare 50, 35 and 20%sucrose solution and keep them at four degrees. Pour the 50%sucrose layer. Freeze it in a minus 40 freezer.
After six hours, add the 35%layer and freeze it in a minus 40 freezer overnight. Pour the first 20%layer and freeze it. On the day of the experiment, remove the necessary number of gradients out of the freezer.
Add the last 20%sucrose layer and let the gradients thaw out in a cold room. Prepare the sample we freshly collected plant. Here, we are using six days'old seedlings of Arabidopsis thaliana.
Harvest the liquid nitrogen frozen plants and grind them thoroughly. Weight 300 milligrams of plant powder. Quickly add 2.4 milliliter of fresh polysome buffer and homogenize.
Pipette the solution into two 1.5 milliliter tubes. Centrifuge the cytosolic extract. Pipette the supernatant.
Be careful to avoid plant fragment. They would spoil the profile in the next step. Load the supernatant on the sucrose gradient without disrupting the gradient.
Put the gradient in an SW 41 rotor bucket.Centrifuge. To visualize the polysome profile and collect the fraction, we use a simple system made of a capillary tube that plunge into the gradient. It is linked to a UV cuvette.
It's self-connected to a peristaltic pump. A UV spectrophotometer registers continuously the OD as the gradient progress through the cuvette. The fraction collector allows collection of two milliliter fractions.
Clean the cuvette and place it in the spectrophotometer. Remove the gradient from the bucket without disturbing it. Chlorophylls have to be concentrated on the top of the gradient.
Plunge the capillary tubes to the bottom of the gradient. Start the peristaltic pump. The gradient is collected from the bottom to the top and OD is continuously read.
Two milliliter fractions are collected. Here is the shape of a classical profile. The RNA precipitation is performed by using guanidinium hydrochloride and isopropanol.
First, pour one volume of guanidinium hydrochloride in a 50 milliliter tube and then add the fraction. Then add 1.5 volume of isopropanol and 50 microgram equal courier. Precipitate the RNA overnight at minus 20 degrees.
Transfer the fractions into 40 milliliter ultra-centrifuge tubes and balance the tube with isopropanol.Centrifuge. Remove the supernatant and air dry the pellet. Resuspend the pellet in 200 microliters of warm TE buffer.
Further extract the RNA by performing a classical phenol chloroform extraction. A representative profile of polysomes extract from Arabidopsis six days'old seedling is shown here. The main peak corresponds to the monosome, the mRNA that are associated with a single ribosome.
The first part of the profile corresponds to the polysomal fractions namely the mRNA associated with many ribosomes. Each peak match the association of the new ribosome to the mRNA. The last part of the profile corresponds to the so called supernatant fraction which contains the free ribosomal subunits and RNA.
Large amount of 60S subunit form a shoulder next to the monosome peak. To assess their integrity, the RNAs extracted from the fractions can be run on a classical Aragose gel before being used for further experiments. We have used this protocol to isolate polysomes from Arabidopsis thaliana seedlings, but also from young and old rosettes as well as from Nicotiana benthamiana, tomato and rice leaves so it should work with a wide range of plants and tissues.
The purified RNAs are compatible with and micro array analysis as well as for the identification of small RNAs associated with polysomal fractions.
