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Isolation and Characterization of Ribonucleic Acid from Yeast

Kristine Bernadette Galvez, Nilufar Haghani Rad, Dixie Mae Lacap, Justyn
Ellin Michaella Laus, Patrick Daniel Libiran
ABSTRACT
Isolation of RNA from yeast, assessment of the purity of the extracted RNA
and lastly the characterization of RNA following basic hydrolysis were the
major objectives of the study. The yeast or scientifically termed as
Saccharomyces cerevisiae was used to isolate RNA. A portion of the
extracted RNA was subjected to alkaline hydrolysis using 0.3 M NaOH. The
RNA hydrolysate was the characterize by four different tests namely test for
ribose, test for phosphate, test for purines or Murexide Test and lastly test for
pyrimidines or Wheeler-Johnson Test. A dark brown solution, yellow
crystalline precipitate, red residue, and a turbid white solution with acidic pH
were obtained from each test respectively. The results from the reaction of
the RNA hydrolysate towards the different tests were compared to the
positive results obtained from the standard RNA sample. Standards served as
the basis whether the extracted RNA sample gave expected results.
INTRODUCTION
Nucleic acids are the main
information carrying molecules of
the cell, they allow organisms to
transfer genetic information from
one generation to the next by
directing the process of protein
synthesis. There are two main
structural classes of nucleic acids
namely deoxyribonucleic acid or
DNA and ribonucleic acid or RNA
which are basically made up of
nitrogenous bases derived from
purines which are adenine (A) and
guanine and from pyrimidines
which are cytosine (C) and uracil
(U), sugar, and phosphate group.
DNA is the master blueprint
for life and constitutes the genetic

material in free-living organisms


and most viruses. RNA is the
genetic material of some viruses,
but it is also found in all living cells,
specifically in ribosomes, where it
plays an important role in certain
process such as the synthesis of
protein.
RNA has a wider range of
functions than the DNA. It is a
biologically important type of
molecule that consists of a long
chain of nucleotide units. If DNA is
usually double-stranded, RNA is
basically a single-stranded nucleic
acid. 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.
According to Campbell-Farrell
there are six kinds of RNA namely
transfer RNA (tRNA), ribosomal
RNA
(rRNA),
messenger
RNA
(mRNA),
small
nuclear
RNA
(snRNA), micro RNA (miRNA), and
small interfering RNA (siRNA).
These various kinds of RNA, in a
series of reactions ultimately
directed by the base sequence of
the cells DNA, participate in the
protein
synthesis.
The
base
sequences of all types of RNA are
determined by that of DNA. And
here is where transcription, the
process by which the order of
bases is passed from DNA to RNA,
will enter.
The RNA was isolated from
yeast or scientifically known as
Sacchararomyces
cerevisae,
a
unicellular fungus that contains 4%
RNA by weight.
The experiment aims first to
isolate RNA from bakers yeast and
then determine the purity of the
extracted RNA by qualitatively
characterize the structure and
composition of RNA following basic
hydrolysis.
METHODOLOGY
Frist the yeast was isolated to
bakers or dry yeast following
hydrolysis and then several test
were performed to test the purity

of the extracted RNA. These tests


are test for ribose, test for
phosphate, test for purines or
Murexide Test and lastly test for
pyrimidines or Wheeler-Johnson
Test.
RNA was isolated from yeast
by water bathing, temperature
adjusted to 60oC, the diluted
mixture of NaOH, water, and dry
yeast for 15 minutes. Centrifuged
and acidified the solution with
glacial acetic acid. And the last
step for the isolation, 95% ethyl
alcohol and concentrated HCl was
added to the supernatant and
stirred vigorously and settled in a
tall covered vessel.
After the isolation, small
amount of the isolated RNA was
mixed with 0.3 M NaOH and water
bath for 60 minutes for alkaline
hydrolysis. The hydrolysate was
cooled and adjusted its pH to pH 46 with glacial acetic acid.
To
have
a
qualitative
characterization several tests were
performed. And standards served
as the basis whether the extracted
RNA sample gave expected results.
First is the test for ribose. A
mixture of hydrolyzed RNA solution
and orcinol reagent was water bath
for 5 to 10 minute. The test was
repeated and standard ribose
solution was used instead.

Second test is the test for


phosphate.
A
mixture
of
concentrated sulfuric acid and
nucleic acid solution was heated
over a small flame and until the
contents of the tube turns brown.
The mixture was cooled and
concentrated nitric acid was added
and was heat again until white
fumes appeared and the solution is
colorless. Water was added and
was heated again for five minutes
in boiling water. And then, cooled
again and 1% (NH4)2MoO4 solution
was added. The test was repeated
and standard phosphate solution
was used instead.
Murexide test or test for
purines is the third test. A mixture
of few drop of nucleic acid solution
and few drops of concentrated
nucleic acid in a small evaporating
dish was evaporated to dryness in
a water bath. And to moisten the
residues 10% KOH were used and
heat further. And lastly few drops
of water was added just for test.
The test was repeated and
standard adenine solution was
used instead.
The last test that was
performed
was
the
WheelerJohnson test or also known as test
for pyrimidines. Excess bromide
water was added into the nucleic
acid solution until the solution
turned yellow. The solution was
boiled then until it turns light
yellow or colorless. Lastly Ba(OH)2

was added and was tested with


litmus
paper.
The
test
was
repeated and standard cytosine
solution was used instead.
RESULTS
Table 1. Experimental Results
Chemical
Test
Test for
Ribose
Test for
Phosphate
Test for
Purines
Test for
Pyrimidin
es

RNA
from
Yeast
darkbrown
solution
yellow
precipitat
e
red
residue
turbid
white
solution
~ acidic

Standard
Solution
dark-blue
solution
yellow
solution
yellow
residue
violet
precipitat
e
~ basic

DISCUSSION
In the test for the presence of
ribose, the orcin reaction was used.
The conversion of ribose to an
aromatic aldehyde which then
reacts with Orcin to form an
aldehyde-phenol
condensation
product that in a theoretical result
is blue-green/dark green in color,
this reaction depends on. The
result we had was a dark brown not
a dark green or blue-green
solution. The failure in getting a
positive in the RNA of yeast was

because the conversion of ribose to


an aromatic aldehyde was not fully
developed.
In the test for the presence of
phosphate in RNA,
a yellow
precipitate is obtained. This is due
to the reaction of ammonium
molybdate solution which when
dropped upon a sample, indicates
the presence of phosphate by a
yellow stain or a crust of yellow
phospho-ammonium molybdate.
In the test for purines, or
commonly known as murexide test,
the RNA is reacted with nitric acid
since purines are known to be
readily soluble in dilute acids.
Concentrated nitric acid oxidized it
leaving a yellow precipitate upon
evaporation. However, it turned red
when moistened with a base, which
is a positive result for presence of
purine bases (guanine or adenine).
In the test for pyrimidines,
the sample is treated with bromine
water
to
form
5-bromo-6hydroxyhydro derivatives which
produces a yellow coloration. Upon
dehydration in solution, it forms a
5-bromo derivative. The addition of

barium hydroxide Ba(OH)2 gives a


5,
5-dibromo-6-hydroxyhydro
derivatives, a violet precipitate,
which is a positive result for the
presence of uracil in RNA.
REFERENCES
Crisostomo,
A.,
et.al.
(2010).
Laboratory Manual in General
Biochemistry. Quezon City: C&E
Publishing Inc., pp. 73-77.
Campbell, M. & Farell, S. (2015).
Biochemistry, 8th Ed. Singapore:
Cengage Learning, p. 239
Roberts, R. (2013). Nucleic Acid.
Retrieved on March 18, 2015 from
http://www.britannica.com/EBcheck
ed/topic/421900/nucleic-acid
Bailey, R. (n.d.). Nucleic Acids.
Retrieved on March 18, 2015 from
http://biology.about.com/od/molecu
larbiology/a/nucleicacids.htm