ISOLATION OF RNA AND UV MEASUREMENT Jenny Lynn A.

Karunungan, Daniel Adam Levy, Josell Mary Engelee Lim, Alphonse Leonard Lomotan, Maria Eloisa Mateos Group6 2B Medical Technology Biochemistry Laboratory ABTRACT
RNA was extracted from Yeast(Saccharomyces cerevisiae) by heating it with alkali NaOH, extracting the nucleic acids and water-soluble proteins as well as inactivates nucleases which degrade the RNA. HCl extraction was used to isolate the RNA from the proteins and lipids were removed by treating with alcohol and ether. The isolated RNAs absorbance was measures at 230 nm, 260 nm and 280nm. The isolated RNA was also characterized using different tests: test for Ribose, Test for Phosphate, Test for Purins(Murexide Test) and Test for Pyrimidines(Wheeler-Johnson Test) was performed, the tests were also done on a hydrolyzed version of the isolated RNA.

INTRODUCTION
Nucleic acids were found to be major components of chromosomes, small genecarrying bodies in the nuclei of complex cells (eukaryotes). They are high molecular weight biopolymers of nucleotides. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).[3] DNA is the one that contains the "programmatic instructions" for cellular activities, when organisms produce offspring, these instructions, in the form of DNA, are passed down. While RNA, it is involved in the synthesis of proteins. "Information" is typically passed from DNA to RNA to the resulting proteins. The difference between the two is based on the nuclear base present, kind of sugar it has, number of strand and process of transferring genetic information; or in simplest term difference in nucleotide, which is the monomer of nucleic acids.[1] The experiment focused mainly on RNA. RNA are single stranded nucleic acids found at high concentration in tissue with large cytoplasmic volume. The high concentration of RNA- mRNA, tRNA, and rRNA in the cytoplasm is due to their functions in relation to protein synthesis. RNA molecules are realatively shorter, and less damaged by shearing during its isolation. They are very vulnerable to digestion by ribonucleases(RNAses) which are present endogenously in various concentration in certain cell types and exogenously on palms and fingers. The most abundant RNA species are the rRNA molecules: 23S and 16S for prokaryotes, and the 28S and 18S for eukaryotes. Eukaryotic mRNA constitutes only 2-5% of the cellular RNA responsible in replication with the DNA and transfer of genetic code for protein synthesis. The smallest of the three major types is the tRNA that contains the amino acids for protein synthesis and complementary nucleocodes for anti-coding with mRNA. Other RNA molecules usually found in eukaryotes are siRNA(small interfering RNA), and microRNA that affect gene expression and are used by the researchers in gene-knockout studies. Isolation of RNA varies according to the type of tissue employed and the particular RNA species to be isolated. The isolation of yeast involves heating with alkali (NaOH) which extracts nucleic acids and water soluble proteins and inactive nucleases; and nucleic acids can then be separated from associated proteins and other interfering substance by acid extraction at ph 4.5. lastly, the final step

is treatment with alcohol and ether to remove lipids. UV spectroscopy is the most widely used for assessing RNA concentration and purity. This is also a widely used technique for quantitative analysis of RNA. The objectives of this experiment is to isolate RNA from yeast, determine the purity of extracted RNA thru spectrophotometric analysis and characterization of RNA following basic hydrolysis.

95% ethanol and twice again with ether. The residue was then air dried and was weighed. The percentage yield obtained was then calculated and the sediment was used for the hydrolysis of nucleic acids.

2. Ultraviolet Measurement of Isolated RNA

A 0.5 mL aliquot of RNA was diluted with 4.5 mL of TE Buffer and was mixed with a vortex mixer. The solution was transferred to a quartz cuvette and the absorbance at 260 nm and 280 nm was determined. Then, the ratio of A260/A280 was calculated and compared to A260/A230 in order to assess the purity of the RNA isolate. Lastly, the total amount of RNA was computed for quantitative analysis.

EXPERIMENTAL A. Compounds tested (or Samples used)

The samples used for this experiment is the RNA isolated form yeast and its alkaline hydrolysate. The RNA isolate appeared as a white precipitate while its alkaline hydrolysate appeared as a clear solution.

3.Alkaline Hydrolysis
2 mL of 0.3M NaOH was added to a pinch amount of RNA isolate. Then, 10 mL of distilled water was added. The test tube containing the solution was then heated in a boiling water bath for 30 minutes making sure that the test tube was covered with cotton balls while it was heated. After 30 minutes, the hydrolysate was cooled and the pH was adjusted to 46 by adding glacial acetic acid.

B.Procedure 1.RNA Isolation form Yeasts

In a beaker, a 5.0 mL of 1% NaOH solution was diluted with 25 mL of water. 1.5 grams of active dry yeast was then added and the solution was heated in a 60C water bath, covered with a watch glass and stirred occasionally. The solution was filtered using a flutted filter paper since it was still hot. The filtrate was then filtered again and an Erlenmeyer flask was used as a receiver. The collected supernatant was made into a slightly acidic mixture by adding drop wise amount of glacial acetic acid. The supernatant was evaporated over a water bath to approximately 10 mL. It was allowed to cool to 40C or lower and then, it was poured, while vigorously stirring, into 20 mL of 95% ethyl alcohol containing 0.2 mL conc HCl.The RNA solution was allowed to settle until the next laboratory period. The setteled particles were then aspirated and the residue was washed twice with 5 mL of

4.Test for Ribose

2 mL of orcinol reagent was added to 0.5 mL of hydrolyzed RNA solution and was mixed in a vortex mixer. The solution was heated in a boiling water bath for 510 minutes and the color of the solution was noted. The same procedure was done using standard ribose solution.

5.Test for Phosphate

1 mL conc H2SO4 was added to 1 mL RNA solution. The mixture was heated in a water bath while shaken frequently until the contents turned brown. The mixture was then cooled before adding 0.5 mL conc HNO3. The mixture was again heated until white fumes appeared. When the mixture became colorless, 1 mL of water

was added and the mixture was heated in a boiling water bath for 5 minutes. The mixture was cooled and 1mL of 10% (NH4)MoO4 solution was added. The mixture was then mixed and was diluted to 10mL of distilled water. The color of the precipitate was noted after letting the mixture stand for 5 minutes. The same procedure was done using hydrolyzed ribose solution and standard phosphate solution.

RESULTS AND DISCUSSION

RNA(Ribonucleic acid) is biological molecule composed of 3 components which are nitrogenous base, five-carbon sugar and phosphate group. RNA contains a monomer, being the Nucleotide and is the main difference of the RNA from the DNA. A nucleotide has 3 components which are nitrogenous base, five-carbon sugar and phosphate group. The nitrogenous base can be classified as pyrimidine or purine which contains aromatic ring/s. The ones classified as pyrimidines are those containing a monocyclic bases while those that are bicyclic bases are purines. It is the one responsible for the determining the genetic identity of an indivual. Pyrimidines are the cytosine(C) and thymine(T), and the purines are the adenine(A) and guanine(G) which can be found for both DNA and RNA except for thymine, found only in DNA and replaced by uracil in RNA. Uracil(U) could be considered a demethylated form of thymine. These nuclear bases produce base pairing through hydrogen bonding. Adenine and thymine/ uracil are connected by two hydrogen bonds while cytosine and guanine have three.

6.Test for Purines (Murexide Test)

5-10 drops of RNA solution was placed in a test tube along with 5 drops of conc. HNO3. The solution was then placed in a water bath until it was evaporated or until a noticeable amount of the solution was evaporated. 5 drops of KOH was then added to the solution and was again heated noting the color of the solution when KOH was added and when the solution was heated. Then, a few drops of water was added and the solution was warmed while noting the color of the solution. Lastly, the solution was allowed to evaporate and again, the color of the residue was noted. The same procedure was applied using hydrolyzed RNA solution and standard adenine solution.

7.Test for Pyrimidines (WheelerJohnson Test) An excess of bromide water was added to 0.5 mL RNA solution until the solution turned yellow. The solution was boiled using a hot plate until it turned light yellow or colorless. Excess Ba(OH)2 was added to the solution to make it basic. Then the color of the solution was noted. The same procedure was applied using the alkaline hydrolysate and standard uracil solution.

Fig.1 Purine bases of RNA and DNA

Fig. 2 Pyrimidine bases of DNA

The RNA aliquot is placed in a cuvette and diluted with TE buffer(pH 8). TE buffer was used to remove the contamination of DNA. Using pure water will provide a ratio close to two, if the solution is pure, but the assumption that the solution is free from DNA contamination cannot be excluded. Results RNA Solution description Turbid Solution A260 0.161 A280 0.215 A230 .208

Fig 3. Structure of Uracil(Replaces Thymine in RNA)

The next component is the five carbon sugar where for DNA it has 2-deoxyribose sugar while RNA has ribose sugar. The difference is the removal of hydroxyl group in the second carbon of DNA sugar. These sugars are connected to the nucleobase pairs thru glycosidic bond forming nucleosides, and two sugars are connected to each other thru phosphodiester linkage with inorganic phosphate to form a polynucleotide.[2] Ultraviolet Measurement of Isolated RNA involved the use of spectrophotometer; this is a quantifying test for RNA. RNA is routinely tested for contaminants at A260 and A280, in the experiment A230, A260 and A280 was tested. The ratio of A260/A280 determines the amount of contaminants inside the RNA solution, a ratio close to 2.0 means that the solution contains no contaminants, or DNA contaminants. Certain contaminants such as phenols are detected at A230, and a ratio lower than 1.8 may indicate the contamination of these contaminants.

The ratios acquired during the experiment showed for very contaminated RNA solution. The A260/280 ratio was 0.751, not nearly close to 2.0 showing a contamination and poorly isolated RNA, the ratio of A260/A230 was 0.774, a ratio far below 1.8 showed the contamination of various molecules. The total RNA in micrograms were then computed or using the following solution
A260 x dilution factor x 40 x sample volume

the total RNA was shown to be 162.5 micrograms. In the Alkaline hydrolysis of RNA, the 2 OH group in ribonucleotides, makes the RNA vulnerable to cleavage in alkali solutions. 0.3M NaOH was added to the RNA isolate and heated. The product is a equimolar 2- and 3-nucleoside monophosphates. The transfer of a phosphate from one nucleotide to an adjacent nucleotide chain was the result.

Chemical Test Test for Ribose Test for Phosphate Test for Purines Test for Pyrimidines

RNA isolate
Yellow to Blue No precipitate or color change Very Pale Yellow Clear with pale yellow ppt

Hydrolyzed RNA
Yellow No precipitate Colorless>yellow>Colorless Colorless Slightly turbid with little white ppt.

Positive Result
Blue-green solution Yellow Crystalline ptt Brown Red residue Purple ppt.

Table. 1 Results of characterization test

The structural features of RNA is tested using characterization tests, with the use of different reagents. The tests may test for the structural features based on reactions. The first test, the test for Ribose was done using the orcinol reagent. Orcinol reagent was added to both Isolated RNA and hydrolyzed RNA. The conversion of ribose to an aromatic aldehyde(Furfural) reacts with Orcin producing an aldehyde-phenol condensation which is responsible for the blue/green color. In the experiment, a positive result was acquired for the unhydrolyzed isolated RNA. However in the Hydrolyzed RNA a negative result was acquired, which may indicate the conversion to aromatic aldehyde was not developed. In the test for Phosphate, the yellow precipitate positive result is due to the reaction of ammonium molybdate the reaction with the phosphate in the RNA. The yellow precipitate is the resulting Phospho-ammonium molybdate. The positive result was not acquired for both isolated RNA and hydrolyzed RNA. This may be because of the lack of HNO3 or the solution was not heated properly. The test for Purines or the Murexide test, basically is the RNA reacting with nitric acid. Purines are readily soluble in dilute acids. Oxidized Nitric acid will leave yellow precipitate when evaporated, then will turn brown red when a base is added which will indicate the presence of the purine bases. A negative result was

acquired for both RNA samples, the refusal of the solution to evaporate when in the water bath was the most likely cause of the result. The final test conducted was the test for Pyrimidines. Also known as the WheelerJohnson Test. The test detects the presence of Uracil in the RNA. Both RNA samples are treated with Bromine water until the solution is yellow, the yellow is because of the formation of 5-bromo-6hydroxyhydro derivatives. Dehydration forms a 5-bromo derivative and the addition of Barium Hydroxide gives the purple precipitate with Uracil. In the experiment the positive result was not acquired, maybe due to the RNA not being isolated properly, or the addition of the reagents was poorly done and judged.

REFERENCES [1]http://biology.about.com/od/molecularbiolo gy/a/nucleicacids.htm [2]http://www.chem4kids.com/files/bio_nuclei cacids.html [3]http://www2.chemistry.msu.edu/faculty/reu sch/VirtTxtJml/nucacids.htm