EXPERIMENT NO. 10 ISOLATION OF RNA FROM YEAST I.

INTRODUCTION Nucleic acids are macro biopolymers of high molecular weight with mononucleotide as the repeating unit. The two structural kinds of nucleic acids are DNA and RNA which are basically made up of nitrogenous bases, sugar, and phosphate group. The sequence of nucleotides allows RNA to encode genetic information. For example, some viruses use RNA instead of DNA as their genetic material. In isolating RNA, heating with alkali, acid extraction, and treatment with alcohol are involved. Yeast, Saccharomyces cerevisiae, is a unicellular fungus that contains 4% RNA by weight; it has low d-ribonuclease and ribonuclease activity and can be readily obtained in essentially pure form from this source. Many proteins in human biology were first discovered by their homologs in yeast. S. cerevisiae was the first eukaryotic genome to be completely sequenced. It was estimated that yeast shares 23% of its genome with humans. The following are the objectives in the experiment entitled, Isolation of RNA from Yeast: To be able to isolate RNA from yeast To be able to get the percentage by mass of RNA from yeast II. MATERIALS AND METHODS In a beaker, 5mL of 1% NaOH solution and 25mL of water were diluted and were added with 3.0g of dry yeast. The mixture was set to heat in a water bath for 15 minutes with occasional stirring. After heating, the mixture was strained with cheesecloth. The filtrate was centrifuged and the supernate was transferred to a different test tube and was added glacial acetic acid dropwise until faintly acidic. It was observed that the supernate was turbid; it was centrifuged and decanted after. 10 mL of 95% ethanol containing conc. HCl was poured to the supernate while it was stirred vigorously. All residues were decanted, centrifuged, and transferred in one big test tube and was washed twice with 1 mL of 95% ethanol. The decantation and centrifugation processes were repeated every after washing. It was washed again twice with small amount of ether with the same process as the previous; it was centrifuged and decanted every after washing. After the washing, the residue was divided into two portions in two separate test tubes and cover. The test tubes were refrigerated to be used in the following experiment in the next laboratory meeting.

III. RESULTS AND DISCUSSION The process of the isolation of RNA from yeast, Saccharomyces cerevisiae, involves heating with NaOH which served to disrupt the cell membrane and lyse the cell to be able to extract the nucleic acids from the said test sample. The NaOH increases the pH level of the solution resulting in the denaturation of contaminant proteins, inactivates nucleases which can degrade RNA. Heating helped loosen the cell membrane by increasing the kinetic energy of the lipid molecules, making it release more RNA. The mixture was filtered and centrifuged to get rid of the denatured proteins, lysed lipid membranes and other contaminants. Centrifugation, which uses the idea of gravity, breaks up the cells and leads to the sedimentation of the largest particles present which is the nuclei. Increasing the centrifugal speed brings about the sedimentation of first the cytoplasmic large and small granules. RNA was obtained from the cytoplasmic fractions. After centrifugation, the supernatant was collected while the residue was discarded. The purpose of the addition of glacial acetic acid was to lower the pH level to help denature more proteins, prevented alkali RNA hydrolysis, ensuring that the desired RNA was not degraded. The mixture was decanted and centrifuged repeatedly to eliminate the precipitated proteins. The reason why the supernate was suggested to be 10mL or below was to increase the RNA in the solution and for it to be easily isolated later on. 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. Centrifugation also separated the RNA precipitate from the unneeded supernatant. Both washings from ethanol and ether removed any lipid residues and other non-polar contaminants. Table1. Percentage by mass of RNA from yeast Mass of yeast, g Mass of RNA, g Percent yield (%) 3.105 0.6324 20.37

After the isolation of RNA, the experimental results showed that 0.6324 g RNA was able to extract from the yeast, Saccharomyces cerevisiae, indicating 20.37% yield from the original sample. Low percentage yield was due to many sources of RNAases brought by the hands. RNases are enzymes whose entire purpose is to degrade RNA. They are very stable proteins that can withstand high temperature and they are everywhere. Once cells the RNAs are broken open, they are susceptible to degradation by RNAses. Thus, wearing gloves during the experiment was needed. . IV. REFERENCES 1.) Elson D (1965). "Metabolism of nucleic acids (macromolecular DNA and RNA)". Annu. Rev. Biochem. 34: 44986 2.) Stryer, L.; Berg, J.; Tymoczko, J. (2007). Biochemistry. San Francisco: W.H. Freeman 3.) Biochemistry Laboratory Manual, Institute of Chemistry, UP Diliman, 2007 Edition. pp.10-15.