Isolation and Characterization of RNA from Saccharomyces cerevisiae

Daniel O. Delos Reyes, Angelica T. Domalanta, Hannah Q. Doria, Isabella C. Guce, Janissa G. Gutierrez Group 4 2B Pharmacy Biochemistry Laboratory

ABSTRACT
Ribonucleic Acid (RNA) was isolated from yeast (Saccharomyces cerevisiae) by heating the active dry yeast with 1% NaOH solution. RNA was then separated from associated proteins with HCl extraction at pH 4.5 and was further treated with ethyl alcohol to remove lipids. To determine the isolated RNA purity, its absorbance was measured at 260 nm and 280 nm that yielded a result of 0.706 and 1.333 which confirmed impurity and presence of contaminants in RNA sample. The RNA was also hydrolysed and characterized by different tests: test for ribose, test for purines (Murexide Test) and test for pyrimidines. It only yielded a positive result for ribose (green solution).

INTRODUCTION
Ribonucleic acid (RNA) molecules are single-stranded polymer nucleic acid [1] made up of ribonucleotides. Each ribonucleotide consists of a ribose sugar, a phosphate, and a nitrogenous base derived from purines which are adenine (A) and guanine (G) and from pyrimidines which are cytosine (C) and uracil (U). In comparison with DNA, RNA is relatively shorter in molecules thus it is not easily damaged by shearing but it is more unstable and more prone to degradation due to having a ribose sugar [2].

for all known forms of life [4]. The three major types of RNA in eukaryotes are involved in essential process of protein synthesis. The mRNA caries the information from the DNA in the nucleus to the site of protein synthesis, tRNA delivers amino acids to the ribosomes and rRNA links amino acids together finally forming the proteins. The objectives of this experiment are the following: to isolate RNA from yeast, to determine its purity with UV spectroscopy, and to characterize RNA with the use of different tests (test for ribose, test for purines, and test for pyrimidines) following a basic hydrolysis. The RNA was isolated from yeast (Saccharomyces cerevisiae), a unicellular fungus that contains 4% RNA by weight. The isolation of RNA from yeast involves heating with NaOH which loosen and lysed the cell membrane resulting in the extraction of RNA. Addition of NaOH and glacial acetic acid to the sample RNA prevents its degradation by increasing its pH level which inactivates the nucleases. In order to get rid of the contaminants such a lipids and to separate the RNA precipitate from unneeded supernatant, the sample RNA was filtered, centrifuged, and washed with ethanol and ether [5]. UV spectroscopy is used to asses concentration and purity by

Figure 1. Ribonucleotide Components RNA is synthesized in the cell by enzymes, RNA polymerase, and involves forming phosphodiester bonds between the 3' carbon of one nucleotide and the 5' carbon of another nucleotide. This leads to formation of the so-called "sugarphosphate backbone", from which the nitrogenous bases project [3]. RNA is one of the three major macromolecules that are essential

RNA

measuring RNAs absorbance at 260 and 280 nm. A highly purified RNA has (absorbance) at the ratio of 1.85-2.0 and presence of organic contaminants such as phenols and other aromatic compound used in the extraction of RNA in the sample lowers the (absorbance) below 1.8 [1]. The test for ribose, for purines (Murexide Test), and for pyrimidines (Wheeler-Johnson Test) are used to determine the presence of ribose, purines, pyrimidines respectively in the sample RNA. In the test for the presence of ribose, the pentose sugar (ribose) is dehydrated to furfural which yields a green solution when reacted with orcinol. The principle behind Murexide Test, used to test the presence of purines, is purine degradation which yields a yellow precipitate and a reddish brown precipitate when treated with base. Lastly, the Wheeler-Johnson Test, used to characterize pyrimidines, yields a dialuric acid, a yellow coloration, when treated with bromine water and yields a purple solution when treated with Ba(OH)2 [6].

with glacial acetic acid. The mixture solution of 20 mL of 95% ethanol and 0.2 mL of conc. HCl was poured to the supernatant and stirred vigorously. All residues were decanted, centrifuged, and washed twice with 2mL of 95% ethanol and with ether. 2. Alkaline Hydrolysis The mixture solution of 2 ml of 0.3N NaOH and isolated RNA was water bath for 60 minutes. The hydrolyzate was cooled and adjusted to pH 4-6 with glacial acetic acid using pH paper. The hydrolysed sample RNA was used for testing. 3. Test for Ribose A mixture of 0.5 ml hydrolyzed RNA solution and 2 ml Orcinol reagent was water bath for 5-10 minutes. The test was repeated and standard ribose solution was used instead. 4. Test for Purines (Murexide Test) Five drops of RNA solution and few drops of concentrated HCL were placed in a small evaporating dish and evaporated to dryness on a hot plate. The residue formed was moistened with 10% KOH and was heated again. Few drops of water were added to the dried solution and were warmed. 5. Test for Pyrimidines (WheelerJohnson Test) Excess bromide water was added to 0.5 ml RNA solution until the solution turned yellow. The solution was boiled until it turned to light yellow or colorless. Excess Barium Hydroxide was added to the solution and was tested with litmus paper.

EXPERIMENTAL
A. Materials The materials used for isolation of yeast were active dry yeast (Saccharomyces cerevisiae), concentrated HCl, glacial acetic acid, 1% NaOH, 95% ethanol and ether. The reagents used for alkaline hydrolysis and characterization of RNA were 0.3N NaOH, 1N KOH, concentrated H2SO4, concentrated HNO3, ammonium molybdate, bromine water, 10% KOH, Ba(OH)2,orcinol reagent, and standard solutions : pentose sugar of RNA (ribose) and nitrogenous bases of RNA (adenine, guanine, cytosine and uracil). B. Procedure 1. RNA Isolation from Yeast The diluted mixture of 5mL of 1% NaOH solution, 25mL of water and 5.0g of dry yeast was water bath for 15 min. at 60 degrees Celcius with occasional stirring. After heating, the mixture was strained with cheesecloth, centrifuged and acidified

RESULTS AND DISCUSSION

1. Ultraviolet Measurement of Isolated RNA In UV measurement of the isolated RNA, it was measured that the absorbance ( ) was 0.706 which means that the RNA was not highly purified. It was also found out that the RNA contains contaminants such as phenol and other

aromatic compounds which lower the samples (absorbance) to 1.333 and could interfere with the analysis and characterization of the RNA sample. Inadequate washing, decanting, filtering and centrifuging of the sample could have cause the isolated RNA to be impure and with contaminants. Dirty cuvettes could also lower the absorbance measured by UV spectroscopy.
Chemical Test Test for Ribose Test for Purines Std. solution Dark Green solution Yellow ppt RNA from yeast Green solution Yellowish brown ppt Brown ppt Litmus: Red to Blue (+) result Green solution Reddish brown ppt

3. Test for Purines (Murexide Test) In the test for purines, or commonly known as murexide test, the RNA from yeast and the standard solution yielded a yellow precipitate when oxidized with nitric acid and evaporated due to purine degradation. However, both turned to turned into reddish brown precipitates when moistened with a base, which is a positive result for presence of purine bases and turned back to yellow ppt (standard solution) and yellowish brown ppt (RNA from yeast) when water was added and evaporated. Prolonged evaporation, inadequate addition of base, and presence of contaminants in the sample might have cause the error.

Test for Pyrimidines

White ppt Litmus: Red to Blue

Purple ppt

Table 1. Chemical Characterization 2. Test for Ribose Positive results for the standard solution (dark green solution) and for the RNA from yeast (green solution) were yielded due to complete conversion of the ribose to an aromatic aldehyde (furfural) which when reacted with Orcinol reagent (3,5-dihydroxy toluene) formed an aldehyde-phenol condensation.

Figure 3 Murexide Test 4. Test for Pyrimidines(WheelerJohnson Test) In the test for pyrimidines, the sample is treated with bromine water to form 5bromo-6-hydroxyhydro derivatives which produces a yellow coloration. Upon dehydration in solution, it forms a 5bromo derivative. The addition of barium hydroxide Ba(OH)2 gives a 5, 5-dibromo6-hydroxyhydro derivatives, a violet precipitate, which is a positive result for the presence of uracil in RNA. Both the standard solution and RNA sample did not yield 5, 5-dibromo-6-hydroxyhydro derivative needed to form a violet precipitate when treated with Ba(OH)2

Figure 2. Reaction of ribose with orcinol reagent .

REFERENCES
[1] Crisostomo, A., et. al. (2010). Labaratory Manaul in Genearal Biochemistry. Quezon City: C&E Publishing Inc., pp. 73-77. [2] Ribonucleic Acid. (n.d.). In Scitable online. Retrieved from http://www.nature.com/scitable/definition /ribonucleic-acid-rna-45 [3] Bowen, R. (2001, Dec 9). The Structure of Nucleic Acids.Retrieved from http://arbl.cvmbs.colostate.edu/hbooks/g enetics/biotech/basics/nastruct.html [4] The RNA Society.(n.d.). What is RNA. Retrieved from http://www.rnasociety.org [5] Elson D (1965). "Metabolism of nucleic acids (macromolecular DNA and RNA)". Annu. Rev. Biochem. 34: 44986 [6] Balda, K. , et. al. (2011) Nucleic Acid RNA [PowerPoint slides]. Retrieved from http://www.slideshare.net/kevbalda/repor t-exp-6-and-7-dna-and-rna