ANALYSIS

The isolation of RNA from yeast involves heating with NaOH which served to disrupt the cell membrane and
lyse the cell extracting the nucleic acids. The NaOH also 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. 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.

Acid Hydrolysis is a chemical process in which acid is used to convert cellulose or starch to sugar. In this
experiment, the RNA yields products of hydrolysis which is the Nucleobases (A,G,U & C), Sugar (D-ribose), and
Phosphate.

The hydrolysate increases amino acid oxidation, it is beneficial for protein synthesis as it reacts quickly to the
reagents and chemical used making the reactions faster than the unhydrolyzed RNA. This was evident in our
observations throughout the experiment when we tested each using the two different RNA. The products in the
Benedicts test both tested positive (+) when they reacted with the hydrolysate, and the unhydrolyzed in separate test
tubes. Orcinol Test or also known as the test for Pentoses only produced one positive (+) product when tested with
the unhydrolyzed; Purine bases yielded an acidic solution in the hydrolysate, while it yielded a basic solution in the
unhydrolyzed. The last experiment conducted was the Inorganic Phosphate test, which produced positive (+)
solutions in both the hydrolysate and unhydrolyzed.

1 Benedicts Test
The Benedicts test, in order to be classified as having positive (+) solutions, should yield colors of green or
red indicating the presence of reducing sugars; The colors that the solutions produced in our experiment were light
green (hydrolysate and unhydrolyzed) indicating the presence of a very low reducing sugar.
 2 Test for Pentoses (Orcinol Test)
Solutions having the presence of pentoses with orcinol reagent (3,5 dihydroxytoluene) will always yield a
blue-green solution. Only one in the experiment produced a positive (+) solution where the unhydrolyzed was
involved; the other solution with the hydrolysate produced a yellow-brown (almost dark) complex, which was due to
prolonged heating of hexoses that yield hydroxymethylfurfurals, which reacts with the orcinol giving the product a
yellow-brown color.

3 Purine Bases
Free purine bases may be separated from the protein nucleosides by precipitation with the help of silver
nitrate (AgNO3). The hydrolysate tested acidic with precipitate (formed quickly) which is a purine complex with
Ag+ ion forms, and the unhydrolyzed that tested as basic with precipitate that was viewed under the microscope.

4 Test for Inorganic Phosphate

There is presence of inorganic phosphate. On warming with the ammonium molybdate in presence of HNO3,
inorganic phosphate is precipitated as yellow ammonium molybdate. Both solutions tested as positive (+) yielding a
yellow color.

The difference between the hydrolysate and the unhydrolyzed RNA was how the reaction takes time;
Reactions that took place with the presence of the hydrolysate were faster than the experiments under unhydrolyzed,
this was due to the fact that the hydrolysate went under acid hydrolysis.

Terms:

Decant- gradually pour (liquid, typically wine or a solution) from one container into another, especially without
disturbing the sediment.

Centrifuge- is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a
potentially strong force perpendicular to the axis of spin (outward).

Supernatant- denoting the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or
other process.
CONCLUSION

Ribonucleic acid (RNA) functions in converting genetic information from genes into the amino acid
sequences of proteins. In this experiment, RNA was isolated from yeast (Saccharomyces cerevisiae) by heating the
active dry yeast with alkaline NaOH. This method of RNA extraction involved the disruption of the cell membrane
and subcellular nucleus to break open and discharge the nucleic acids. RNA was extracted from associated proteins
with HCl extraction and was treated with ethanol and ether to remove lipids.

REFERENCES

http://www.pocdscientifc.com.au
http://www.slideshare.net/mobile/kevbalda/report-exp-6-and-7-dna-and-rna
http://publications.lib.chalmers.se/records/fulltext/166692.pdf
http://www.nou.edu.ng/uploads/NOUN_OCL/pdf/pdf2/BIO%20218.pdf
http://umanitoba.ca/Biology/BIOL1020/lab2/biolab2_2.html
http://www.jbc.org/content/159/1/211.full.pdf