Isolation and Characterization of RNA

Evans Jarrel C. Dion, Joy Emmari Diane B. Elguira, Patricia C. Esteban,

Tyrone Jay T. Feliciano, Joshua Hadrian G. Fernandez, Joan Nicole D. Funelas
Group 4 2D-Medical Technology 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 0.876 and 0.491 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 singlestranded polymer nucleic acid
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.

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.
The RNA was isolated from yeast, 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.
UV spectroscopy is used to asses RNA
concentration and purity by measuring RNAs
absorbance at 260 and 280 nm. A highly purified
RNA has

Ribonucleotide Components

Figure 1.

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 socalled "sugar-phosphate backbone", from which
the nitrogenous bases project.
RNA
is
one
of
the
three
major macromolecules that are essential for all
known forms of life. 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.

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 in the sample RNA. In the
test for the presence of ribose, the pentose sugar
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 WheelerJohnson 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.

added to the solution and was tested with litmus

paper.

EXPERIMENTAL

RESULTS AND DISCUSSION

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 3.0g of dry yeast
was water bath for 15 min. at 60 degrees Celsius
with occasional stirring. 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
isolated RNA was water bath for an hour.
hydrolyzate was cooled and adjusted to pH
by using glacial acetic acid using pH paper.
hydrolysed sample RNA was used for testing.

and
The
4-6
The

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 to ten 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 bromine 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

1.

was

Ultraviolet Measurement of Isolated

RNA

In UV measurement of the isolated RNA, it

measured
that
the
absorbance
(

A 260/ A 280 ) (0.876/0.491) was 1.78 which

means that the RNA was not highly purified. A

total RNA of 175.2 micrograms was obtained
from the isolation.

Chemical
Test

Std.
solution

RNA
from
yeast

(+)
result

Test for
Ribose

Dark
Green
solution

Green
solution

Green
solution

Test for
Purines

Yellow ppt

Yellowish
brown
ppt

Reddish
brown
ppt

Test for
Pyrimidine
s

White ppt
Litmus:
Red to
Blue

Brown
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 2.
reagent .

Reaction

of

ribose

with

orcinol

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.

REFERENCES
[1]
Crisostomo, A., et. al. (2010). Labaratory
Manaul in Genearal Biochemistry. Quezon City:
C&E Publishing Inc., pp. 73-77.
[2] Balda, K. , et. al. (2011) Nucleic Acid RNA
[PowerPoint
slides].
Retrieved
from

Figure 3 Murexide Test

4. Test
for
Pyrimidines(WheelerJohnson Test)
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-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-6hydroxyhydro derivative needed to form a violet
precipitate when treated with Ba(OH)2

http://www.slideshare.net/kevbalda/report-exp6-and-7-dna-and-rna
[3] Ribonucleic Acid. (n.d.). In Scitable online.
Retrieved
from
http://www.nature.com/scitable/definition/ribonu
cleic-acid-rna-45