RNA Isolation from Yeasts

RNA was extracted from active dry yeast. This was done by heating active dry yeast, 1% sodium hydroxide, and distilled water mixture in a 60°C water bath for 15 minutes. Heating of the yeast mixture was done in order to disrupt the cell membrane and lyse the cell extracting the nucleic acids. Because of the high level in pH, denaturation of contaminant protein occurs. Contaminant proteins can inactivate nucleases which can degrade RNA. Heat was used to help in loosening the cell membrane by increasing kinetic the kinetic energy of the lipid molecules in the cell membrane which causes the release of more RNA. The appearance of the mixture was creamy whitish-yellowing liquid. This was strained through a cheesecloth. The mixture was now put in a centrifuge (10,000 rpm for 5 mins). This was done to separate the denatured proteins, lysed lipid membranes and other contaminants from the RNA. The supernatant was taken and glacial acetic acid was added to it in order to lower the pH level to help in denaturing more proteins, and to prevent alkali RNA hydrolysis, to ensure that the desire RNA was not degraded. The solution was centrifuged again (10,000 rpm for 5 mins) and the supernatant was taken and 0.2 mL concentrated HCl and 95% ethyl alcohol was added to the solution. Ethanol was added to lower the dielectric constant of the solution and reduced the solubility of RNA causing it to precipitate from solution. HCl was added to protonate the phosphate groups in nucleic acid backbones, minimizing the charge repulsions between molecules and helped aggregate and precipitate. The solution was stored in the locker to let the RNA settle down. The supernatant was removed for this experiment. The residue was washed twice with 95% ethanol and twice with ether.