The overall goal of the following experiment is to identify translating mRNAs in primary CNS tissue. The protocol starts with extracting the relevant tissues from mouse in cycloheximide. This is followed by mechanical homogenization and lysis in detergent.
Next myelin flotation is performed, which removes the fatty lipid components of the tissues that will mask the poly ribosome profiles. The pellet, which contains ribosomes and associated mRNAs, is sedimented on a sucrose gradient to separate mRNA associated with ribosomes. The gradient is then fractioned to provide a poly ribosome profile with each peak representing mRNAs associated with a certain number of ribosomes or ribosomal subunits.
Fractions containing poly ribosomes are then purified obtaining RNA for downstream applications. The main advantage of this technique of existing methods is that it allows working with low amounts of CNS tissue and it's versatile in its downstream applications. This method can help answer key questions in the study of translation in the nervous system, such as changes in translation levels in different conditions, and the role of translation, regulatory factors, and sequence motives.
Generally speaking, individuals new to this method will struggle because in vivo CNS materials are usually limited, and the atic components present in the tissue may mask the poly ribosome profiles To a mouse that was trans cordially perfused with HBSS, with cyclo Heide, quickly start the tissue extraction. Dorsally open the skull and remove the brain. Transfer the brain to ice cold buffer A and directly dice the brain tissue.
Next, perform a laminectomy of the thoracic and lumbar vertebral column. Remove four centimeters of spinal cord and transfer to ice cold buffer A on ice. Dice up the separate tissues to increase their uptake of cyclo heide, and then incubate on ice for 15 minutes.
Next, disrupt the tissues with the down homogenizer to ensure the nuclei remain intact. Brain tissue requires five strokes with a tight pestle, transfer the mesh tissue into tubes. Spinal cord tissue requires five strokes with a loose pestle, and then another five strokes with a tight pestle.
Now collect aliquots for total RNA isolation, 200 microliters of spinal cord and 400 microliters of brain flash. Freeze them and store them at minus 80 degrees Celsius. To remove the nuclei undisrupted cell and tissue fragments, centrifuge the tissues at 500 Gs for 10 minutes.
Collect the supernatant containing the endoplasmic reticulum with its bound ribosomes, and discard the pellet containing the nuclei. Next, lyce the endoplasmic reticulum membrane fragments to release the ribosomes by adding NP 40 and sodium deoxy coate detergents each to a final concentration of 1%Allow the samples to incubate for 30 minutes on ice. One of the challenges of working with nervous system tissues is that myelin and other lipid rich components present with Master Polysome profiles.
In order to tackle this problem, the process of modeling flotation has to be introduced to remove these components Before starting. Pre chilled the ultracentrifuge buckets and rotor at four degree Celsius. Now mix the collected lysates with 1.22 volumes of two molar sucrose.
Transfer each mix to a polyethylene ultracentrifuge tube and fill the tubes to 10 milliliters with 1.1 molar sucrose. Then carefully overlay the tubes with 0.9 molar sucrose. Now transfer the tubes to the chilled buckets and balance them with buffer B.Load the balanced buckets into the chilled rotor and run the centrifuge at 100, 000 Gs.For three hours at four degrees Celsius, the ribosomes and RNAs will pellet under suspended lipids.
After the myelin flotation spin, remove the supra natin and dissolve the pellets with ribosomes in buffer B.Then measure the absorbance of the ribosome solutions at 260 nanometers using a NanoDrop apparatus. Estimate the total RNA concentrations with these values. Now transfer the samples onto prepared sucrose.Gradients.
Take care not to disrupt the gradients and include an empty gradient as a control. Balance the chilled buckets with the samples as before. Then run the centrifuge at 285, 000 Gs for one and a half hours at four degrees Celsius.
While the centrifuge is spinning, prepare the fractionator. First, set the UV lamp sensitivity. Next, attach the tube piercer to the fractionator and connect it via a tube to a bottle of 60%sucrose.
With an intervening rolling pump, start pumping the sucrose into the fractionator at one milliliter per minute. Check for any air leaks that introduce bubbles and tighten up those seals. Then manually pump gradient buffer into the UV detector to set the baseline.
After carefully unloading the samples from the ultracentrifuge without any bumping load one into the fractionator, then pierce the bottom of the tube with the piercer. Start pumping the 60%sucrose to displace the gradient past the UV detector and into the drop dispenser. Record the absorbance profile at 260 nanometers.
Collect 20 fractions from the drop dispenser into two milliliter tubes. Collect 600 microliters per fraction and proceed with processing the sample gradients to each fraction at 10%SDS to a final concentration of 1%SDS and mix well. Now isolate the RNA using an acidic phenyl chloroform extraction.
Begin with adding one volume of room temperature, acidic phenyl chloroform, and then heat the tubes to 65 degrees for 10 minutes. Carefully open the vials in a fume hood after heating to release the pressure. Then centrifuge the fractions at 17, 000 GS for 20 minutes at room temperature and collect the aqueous phase, which may be above or below the sucrose.
Next, add one volume of isopropanol, one ninth volume of sodium acetate, and one microliter of glyco blue. Precipitate the RNA for at least an hour at minus 80 degrees, and then pellet it by centrifugation. Then wash the pellets once with chilled 80%ethanol and after pelleting the RNA, again, air dry the pellets, resuspend them in water and proceed with downstream analysis.
Alternatively, samples can be stored at minus 80 degrees until usage. A blank sucrose gradient shows some background absorption at 260 nanometers due to the presence of DTT. This background can be subtracted during data analysis in order to normalize sample values, the profiles of brain and spinal cord, RNA show characteristic absorption curves mRNAs bound to small ribosomal subunits sediment at lighter fractions coming early on the profile.
They are followed by the large ribosomal subunit and mono bound mRNAs mRNAs bound to multiple ribosomes sediment at later fractions. Treatment of the samples with EDTA collapsed the poly ribosome peaks demonstrating that the sedimentation profile was due to translation. Additional methods like Ribot attack and trap can be coupled to this technique in order to perform cell type specific studies on translation.
After this development, this technique paved the way for researchers in the field of neurobiology to explore the role of translation regulation in neurono development and diseases. After watching this video, you should have a good understanding of how to perform polysome fractionation from low amounts of CNS tissue and purify RNA for subsequent downstream applications of your choice.
