ISOLATION AND CHARACTERIZATION OF RNA FROM YEAST

Lalog, Adrian D., Manalo, Micah M., Manipol, Erwin T., Mendoza, Hannah Daphne M., Mojica, Victoria
Leonesse O.
Group 5 2B BIOCHEMISTRY BIOCHEMISTRY LABORATORY

ABSTRACT
Ribonucleic acid or RNA is one of the three major biological macromolecules that are essential for all
known forms of life. It plays a major role in protein synthesis as it is involved in the transcription,
decoding, and translation of the genetic code to produce proteins. [2] The isolation of RNA from yeast was
done by extracting the nucleic acids and soluble proteins through heating the sample with NaOH. These
nucleic acids were then separated from associated proteins by acid extraction at a pH around 4-6.
Afterwards, it was treated with ether and alcohol to isolate the RNA. The isolated RNA was subjected to
different characterization tests such as UV spectroscopy, Test for Ribose, Test for Phosphate, Murexide
Test, and Wheeler-Johnson Test. For the UV measurement, the absorbance are 0.866, 0.992, 0.969 at
230, 260, and 280nm respectively; while for the different characterization tests, a standard was used to
compare the positive results of each test to the sample.

INTRODUCTION
RNA (Ribonucleic acid) is a polymeric substance
present in living cells and many viruses,
consisting of a long single-stranded chain of
phosphate and ribose units with the nitrogen
bases adenine, guanine, cytosine, and uracil,
which are bonded to the ribose sugar. RNA is
used in all the steps of protein synthesis in all
living cells and carries the genetic information for
many viruses.[1]
The isolation of RNA varies according to the
type of tissue employed and the particular RNA
species to be isolated. The isolation of RNA from
yeast involves heating with alkali (NaOH) which
extracts nucleic acids and water soluble proteins
and inactivates nucleases which can degrade
RNA.
The traditional method for assessing RNA
concentration and purity is UV spectroscopy. The
absorbance of a diluted RNA sample is measured
at 260 and 280 nm. The nucleic acid
concentration is calculated using the BeerLambert law, which predicts a linear change in
absorbance with concentration.[3]

A=lc
Where,
A= absorbance at a particular wavelength
= the extinction coefficient

l = path length of the cuvette

c = concentration of nucleic acid

Using this equation, an A260 reading of 1.0 is

equivalent to ~40g/mL single-stranded RNA.
The A260/A280 ratio is used to assess RNA purity.
An A260/A280 ratio of 1.82.1 indicates highly
purified RNA and an A260/A230 ratio lower than 1.8

indicates the presence of organic contaminants

that may interfere with downstream processes
and analysis.
UV spectroscopy is the most widely used
method to quantitate RNA. It is simple to
perform, and UV spectrophotometers are
available in most laboratories.
The aim of this experiment is to isolate RNA
from yeast, identify the purity of the extracted
RNA and to characterize RNA following basic
hydrolysis.

MATERIAL AND METHODS

The reagents used in this experiment were
0.3M NaOH, 10% KOH, concentrated H2SO4,
concentrated HNO3, concentrated HCl, Ba(OH)2,
10% (NH4)2MoO4 solution, orcinol reagent,
bromine water, glacial acetic acid, 95% ethanol,
ether, and TE buffer.
Also, standard solutions of ribose, guanine, and
cytosine were used for the characterization tests.

A. Isolation of RNA from yeast

In a beaker, 3.0g of active dry yeast was added
to a mixture of 5.0mL of 1% NaOH solution and
25mL of distilled water. The mixture was placed
in a 60C water bath for about fifteen minutes,
cooled and then filtered using cheesecloth. The
filtrate was then added with glacial acetic acid
and was evaporated in a water bath to
approximately 10 mL. After cooling, it was
poured in 20mL of 95% ethyl alcohol containing
0.2mL of concentrated HCl. The RNA was allowed
to settle in a hard test tube inside the fridge for
about two days. The residue in the upper layer of
the mixture was decanted and washed twice with
5mL of 95% ethanol and ether.

B. Ultraviolet
Isolated RNA

Measurement

of

A 0.5mL aliquot of the RNA isolate was diluted

with 4.5mL TE buffer. The solution was then
transferred to a cuvette and the absorbance at
260nm and 280nm was determined.

C. Alkaline Hydrolysis
A small amount of the RNA isolate was placed
in a test tube containing 2mL 0.3 M NaOH. The
mixture was heated in a boiling water bath for
about one hour with paraffin film as cover.
Afterwards, hydrolysate was cooled and adjusted
to a pH around 4-6 with glacial acetic acid.

D. Characterization of RNA
a. Test for Ribose
About 0.5mL of the standard ribose solution
was mixed with 2mL of the orcinol reagent. It
was then placed in a boiling water bath and the
resulting change in color was noted.

b. Test for Phosphate

One milliliter of concentrated H 2SO4 was added
to 1mL of a standard phosphate solution and was
heated in a water bath until the contents turned
brown. After cooling, 0.5mL of HNO 3 was added
and heated again until the appearance of white
fumes. One milliliter of distilled water is added
and the mixture was heated again for additional
five
minutes.
Afterwards,
1mL
of
10%
(NH4)2MoO4 and 10mL of distilled water was
added and the mixture was allowed to stand for a
while and the color of the precipitate formed was
noted.

c. Test for Purines (Murexide Test)

An evaporating dish with 10 drops of Guanine
solution and a few drops of HNO 3 was placed in a
hot plate until dry. The residue formed in the
evaporating dish was moistened with 10% KOH
and was heated again. After drying, few drops of
distilled water were added and the solution of the
solution was noted. It was then dried again and
the residue formed was also noted.

d. Test for Pyrimidines (WheelerJohnson Test)

About 0.5mL of cytosine solution was treated
with excess bromine water until the solution
turned yellow and was boiled until colorless.
Afterwards, excess Ba(OH)2 solution was added.
It was tested with litmus paper and the color of
the solution was noted.

RESULTS AND DISCUSSION

After heating the yeast with alkali (NaOH),
repeated heating and separation of the RNA
isolate to associated proteins and interfering
substances by acid extraction using alcohol, the
RNA precipitated out. The RNA isolate was then
subjected to quantitation using UV spectroscopy
to assess the purity and concentration of the
RNA.
Table 1. Absorbance of the RNA isolate

Sample

A230

A260

A280

RNA isolate from

yeast

0.866

0.992

0.969

Table 1 shows the absorbance of the RNA

isolate at different wavelengths. These were then
used to compute for the A260/A280 ratio and the
A260/A230 ratio to see if the RNA isolate was highly
purified or if there were contaminants present.
Computation:
A260/A280= 0.992/0.969
= 1.02
A260/A230= 0.992/0.866
= 1.15
In the computations shown above, an
absorbance ratio of 1.02 between wavelengths
260nm and 280nm may indicate that the isolated
RNA was not pure.
Also, an absorbance ratio of 1.15 between
wavelengths 260nm and 230nm proves that
there are organic contaminants or impurities
present in the RNA isolate.
Hence, the result indicates that the isolated
RNA isolate contains impurities alongside with
RNA.
Table 2. Characterization Tests for RNA
Chemical Test

(+) Visible Result

Test for Ribose

Dark Green Solution

Test for Phosphates

Yellow Crystalline
precipitate

Test for Purines

Reddish Brown Residue

Test for Pyrimidines

Violet Precipitate

Table 2 shows the positive visible results of the

tests. This was done using the standards
solutions to incur a positive result.

Test for Ribose

Test for Pyrimidines

Bromine water reacts with the sample to form
5-bromo-6hydroxyhydroxo derivative which upon
the addition of Ba(OH)2 give a purple precipitate.
The standard Cytosine solution gave a positive
result of purple precipitate.

Test for Ribose is a test for pentose or a 5carbon sugar. A pentose will be dehydrated to
form furfural which then reacts with the orcinol to
generate a colored substance (dark blue green)
which was observed in the experiment using the
standard ribose solution.

Test for Phosphate

A yellow precipitate should be obtained if
phosphate is present. This is due to the reaction
of the ammonium molybdate solution producing
phosphoammonium molybdate which is a yellow
crystalline precipitate. However, in the
experiment, no yellow precipitate was formed.
This may be because of the contamination during
the preparation of the standard phosphate
solution.

Test for Purines

Guanine is an example of a purine and we
know that purines are readily soluble in dilute
acid. The nitric acid oxidizes it leaving a yellow
precipitate upon evaporation. In the experiment,
only a very light negligible yellow residue was left
in the evaporating dish. This may be because of
possible contamination of the standard solution
used.

REFERENCES:
[1] vlab.amrita.edu,. (2011). Isolation of RNA.
Retrieved 18 April 2016, from vlab.amrita.edu/?
sub=3&brch=186&sim=718&cnt=1
[2] The RNA Society. What is RNA?. Retrieved
April 18, 2016, from
http://www.rnasociety.org/about/what-is-rna/
[3] Wilfinger WW, Mackey K, and Chomczynski P
(1997) Effect of pH and ionic strength on the
spectrophotometric assessment of nucleic acid
purity. Biotechniques 22:474481
[4] Buenafe, R.J. (2013). Test for Carbohydrates
(Bial and Iodine). Retrieved April 18, 2016, from
https://prezi.com/vfjrplptge8b/test-forcarbohydrates-bial-and-iodine/