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 for all known forms of life [4]. The three

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].
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
Figure 1. Ribonucleotide Components glacial acetic acid to the sample RNA
prevents its degradation by increasing its
RNA is synthesized in the cell by pH level which inactivates the nucleases.
enzymes, RNA polymerase, and involves In order to get rid of the contaminants
forming phosphodiester bonds between such a lipids and to separate the RNA
the 3' carbon of one nucleotide and the 5' precipitate from unneeded supernatant,
carbon of another nucleotide. This leads to the sample RNA was filtered, centrifuged,
formation of the so-called "sugar-
and washed with ethanol and ether [5].
phosphate backbone", from which the
nitrogenous bases project [3].
UV spectroscopy is used to asses
RNA is one of the three RNA concentration and purity by
major macromolecules that are essential measuring RNA’s absorbance at 260 and

This study source was downloaded by 100000857575723 from CourseHero.com on 11-27-2022 19:30:20 GMT -06:00

https://www.coursehero.com/file/20764537/203962911-Isolation-and-Characterization-of-Nucleic-Acid/
 280 nm. A highly purified
A 260/ A 280
(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
A 260/ A 230
(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].
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.
This study source was downloaded by 100000857575723 from CourseHero.com on 11-27-2022 19:30:20 GMT -06:00
https://www.coursehero.com/file/20764537/203962911-Isolation-and-Characterization-of-Nucleic-Acid/
RNA has After heating, the mixture was strained
with cheesecloth, centrifuged and acidified
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 (Wheeler-
Johnson 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.
RESULTS AND DISCUSSION
1. Ultraviolet Measurement of
Isolated RNA
In UV measurement of the isolated
RNA, it was measured that the absorbance
( A 260/ A 280 ) 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 Figure 2. Reaction of ribose with orcinol
A 260/ A 230 reagent .
sample’s (absorbance) to
1.333 and could interfere with the analysis 3. Test for Purines (Murexide
and characterization of the RNA sample. Test)
Inadequate washing, decanting, filtering In the test for purines, or commonly
and centrifuging of the sample could have known as murexide test, the RNA from
cause the isolated RNA to be impure and yeast and the standard solution yielded a
with contaminants. Dirty cuvettes could yellow precipitate when oxidized with
also lower the absorbance measured by nitric acid and evaporated due to purine
UV spectroscopy. degradation. However, both turned to
turned into reddish brown precipitates
when moistened with a base, which is a
RNA positive result for presence of purine
Chemical Std. (+)
from bases and turned back to yellow ppt
Test solution result
yeast (standard solution) and yellowish brown
Dark ppt (RNA from yeast) when water was
Test for Green Green added and evaporated. Prolonged
Green
Ribose solution solution evaporation, inadequate addition of base,
solution
and presence of contaminants in the
Yellowish Reddish
Test for sample might have cause the error.
Yellow ppt brown brown
Purines
ppt ppt

White ppt Brown

Test for
Litmus: ppt Purple
Pyrimidine
Red to Litmus: ppt
s
Blue Red to
Blue
Table 1. Chemical Characterization

2. Test for Ribose

Positive results for the standard
solution (dark green solution) and for Figure 3 Murexide Test
the RNA from yeast (green solution)
were yielded due to complete 4. Test for Pyrimidines(Wheeler-
conversion of the ribose to an Johnson Test)
aromatic aldehyde (furfural) which In the test for pyrimidines, the sample is
when reacted with Orcinol reagent treated with bromine water to form 5-
(3,5-dihydroxy toluene) formed an bromo-6-hydroxyhydro derivatives which
aldehyde-phenol condensation. 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. 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

REFERENCES
[1] Crisostomo, A., et. al. (2010).
Labaratory Manaul in Genearal

This study source was downloaded by 100000857575723 from CourseHero.com on 11-27-2022 19:30:20 GMT -06:00

https://www.coursehero.com/file/20764537/203962911-Isolation-and-Characterization-of-Nucleic-Acid/
 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/r
ibonucleic-acid-rna-45

[3] Bowen, R. (2001, Dec 9). The Structure

of Nucleic Acids.Retrieved from
http://arbl.cvmbs.colostate.edu/hbooks/ge
netics/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: 449–86

[6] Balda, K. , et. al. (2011) Nucleic Acid

RNA [PowerPoint slides]. Retrieved from
http://www.slideshare.net/kevbalda/report-
exp-6-and-7-dna-and-rna

This study source was downloaded by 100000857575723 from CourseHero.com on 11-27-2022 19:30:20 GMT -06:00

https://www.coursehero.com/file/20764537/203962911-Isolation-and-Characterization-of-Nucleic-Acid/
Powered by TCPDF (www.tcpdf.org)