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 60C 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.