ISOLATION OF RNA AND UV MEASUREMENT

Sharika Mae Espineli, Kamilah Fernando, Chester Bill Galapon,

Jeana May Galinato and Jairish Keith Garcia
Group 4 2F Medical Technology Biochemistry Laboratory

ABSTRACT
Ribonucleic acid (RNA) was extracted from yeast (Saccharomyces cerevisiae) by heating it
in a water bath with alkali NaOH, extracting the nucleic acids and water-soluble proteins as
well as the inactivating nucleases which degrade the RNA. Hydrochloric acid extraction was
used to isolate RNA from the proteins and lipids were removed by treating with alcohol and
ether. The isolated RNAs absorbance was measured at 260 nm and 280 nm. The isolated
RNA was also characterized using different tests: test for Ribose, test for Phosphate, test
for Purines and test for Pyrimidines. These tests were done on a hydrolyzed isolated RNA.

INTRODUCTION
Ribonucleic acid (RNA) molecules
are single-stranded polymer nucleic
acid made up of ribonucleotides [1].
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)
[2]. They differ in the second major
pyrimidine base that binds with
adenine, thymine in DNA and uracil
in RNA. Another major difference
between DNA and RNA is their sugar
components. DNA lacks a hydroxyl
group attached to the pentose ring in
the 2 position, which makes RNA
less stable than DNA because RNA is
more prone to hydrolysis [3].
Like proteins, some RNA molecules
play an active role in cells by
catalyzing
biological
reactions,
controlling gene expression, or
sensing
and
communicating
responses to cellular signals. One of
these active processes is protein
synthesis,
a
universal
function
whereby mRNA molecules direct the
assembly of proteins on ribosomes.
This process uses transfer RNA
(tRNA) molecules to deliver amino
acids to the ribosome, where
ribosomal RNA (rRNA) links amino
acids together to form proteins [4].
The objectives of this experiment
are to isolate RNA from yeast where
it can be assessed of its purity with
UV measurement and to characterize
and identify the principle involved in
the reactions of RNA with different
tests (test for ribose, test for

phosphate, test for purines, and test

for pyrimidines) following a basic
hydrolysis.
EXPERIMENTAL
A Compounds Tested
The materials used in this
experiments were Active dry yeast,
1% NaOH, Glacial acetic acid, Conc.
HCl, 95% ethanol, Ether, and Litmus
Paper.
B. Procedure
1. RNA Isolation from Yeasts
A 25 mL water was
diluted with 5.0 mL 1% NaOH
solution. A 3.0 g active dry yeast was
added to the diluted solution and was
heated at 60 degrees Celsius with
stirring occasionally on the solution
for 15 minutes. Using a cheesecloth,
the solution was strained. The
solution was then put in a centrifuge
machine. The glacial acetic acid was
added to the supernatant after it was
cooled down to make the solution a
slightly
cidic
mixture.
The
supernatant was evaporated over a
water bath to approximately 10 mL.
It was allowed to cool to 40 degrees
celsius and then a 95% ethyl alcohol
containing 0.2 mL conc. HCl was
poured into the solution while
vigorously stirring the supernatant.
The supernatant was allowed to
settle in order to get the RNA. The
RNA was decanted and was washed
with a 5mL 95% ethanol and wither
twice. The residue was then air dried
and was weighed. The percentage
yield was then computed and the

sediment was used for the hydrolysis

of nucleic acid.
2. Ultraviolet Measurement of
Isolated RNA
A 0.5 mL aliquot with
4.5 mL buffer was used to dilute the
aliquot of the RNA. The solution was
transferred to a quartz coveter and
the absorbance at 260 nm and at
280 nm was determined. The
A260/A280 was calculated and was
compared to A260/A230 to asses the
purity of the RNA isolated. Lastly, the
total RNA was calculated using the
following formula: Total RNA (g) =
A260 x dilution factor x 40 x sample
volume.
RESULTS AND DISCUSSION
A spectrophotometer was used to
determine the concentration of RNA
present in the mixture, as well as its
purity. This is considered as a
quantifying test for RNA. RNA
measurement
is
conducted
by
measuring the ultraviolet absorbance
at 260 nm and 280 nm. RNA absorbs
UV light very efficiently making it
possible to detect and quantify either
at concentrations as low as 2.5
ng/l. The nitrogenous bases in
nucleotides have an absorption
maximum at about 260 nm. RNA
purity is based on the 260 nm/280
nm ratio. It determines the amount
of contaminants inside the RNA
solution. A ratio close to 2.0 means
that the solution contains no
contaminants. However, a low ratio
indicates contamination by protein.
An aliquot of the RNA is diluted
with TE buffer and placed in a
cuvette. TE buffer was used to
remove the contamination of DNA. A
ratio close to two can be inferred
that the solution is pure, but the
assumption that the sample is free of
contamination cannot be omitted.
For nucleic acids, three main
wavelengths of interest are 260 nm,
280 nm and 230 nm. Absorbance at
260 nm is used to measure the
concentration or the amount of

nucleic acid that is present in the

solution. Measurements at 230 nm
are used to determine the amount of
contaminants that may be present in
the sample.
Table 1. Results from the Ultraviolet
Measurement of Isolated RNA
Results
RNA

Solution
descriptio
n
Turbid
solution

A260

A280

A230

0.161

0.215

0.208

The ratios that were obtained

based on the results showed that the
RNA solution was very contaminated.
The A260/280 ratio computed was
0.749, showing a poorly isolated
RNA. It also showed contamination.
The ratio A260/A230 was 0.774, a
ratio
below
1.8
showed
contamination of different molecules.
There were several tests that were
conducted
for
the
chemical
characterization of RNA. In alkaline
hydrolysis of RNA, the 2OH group in
ribonucleotides, makes the RNA
vulnerable to cleavage in alkali
solutions. Nucleic acids are all held
together by phosphodiester bonds.
These
ester
bonds
are
also
hydrolysed with consumption of the
alkali,
rapidly
destroying
the
ribonucleic acid (RNA) [1]. In an
alkaline solution, OH- ions in the
solution remove a proton from the
2-OH of ribose; the 2-O- then is
attracted to the central, relatively
positive phosphorus. The resulting
intermediate can be resolved by the
cleavage of the phosphodiester bond
and the breaking off of the next
nucleotide in the chain [2].
The Test for Ribose was conducted
using the orcinol reagent. The orcinol
reagent was added to both the
hydrolysed RNA solution and the
standard
ribose
solution.
The
conversion of ribose to an aromatic
aldehyde reacts with Orcin producing
an aldehyde-phenol condensation,
responsible for the blue-green/dark
green color of the solution.

The Test for Phosphate is a test for

the presence of phosphate in RNA. A
positive result is the production of
yellow precipitate. This is because of
the
reaction
of
ammonium
molybdate which when dropped upon
the sample, showed the presence of
phosphate by a yellow stain or a
crust of yellow phospho-ammonium
molybdate.
The Test for Purines is also known
as Murexide Test, the RNA is reacted
with concentrated nitric acid. Purines
are known to be readily soluble in
dilute acids. The nitric acid oxidized it
leaving a yellow precipitate when
evaporated. A base is added that will
turn the yellow precipitate to a
brown red residue indicating the
presence of purine bases.
The Test for Pyrimidines, also
known as the Wheeler-Johnson Test
detects the presence of Uracil in the
RNA solution. The sample was
treated with Bromine water until the
solution becomes yellow. The yellow
solution shows the formation of 5bromo-6-hydroxyhydro derivatives.
Upon dehydration, the solution forms
a 5-bromo derivative. A violet
precipitate indicates a positive result

for the presence of uracil in RNA due

to the addition barium hydroxide,
giving a 5,5-dibromo-6hydroxhydro
derivatives
which
is
a
violet
precipitate.
GENERALIZATION
RNA is a single stranded nucleic
acid which yields positive results in
the test for ribose, test for
phosphate, test for purines, and test
for pyrimidines due to the presence
of ribose, phosphate group, purines
(adenine
and
guanine),
and
pyrimidimes (uracil and cytosine).
REFERENCES
[1]http://www.newsmedical.net/health/What-isRNA.aspx (Retrieved March
2015)

18

[2]:http://biology.about.com/od/mol
ecularbiology/ss/rna.htm (Retrieved
March
18
,
2015)
[3]http://www.bibliotecapleyades.net
/vida_alien/esp_vida_alien_18m1.ht
m (Retrieved March 18 , 2015)
[4]http://www.ncbi.nlm.nih.gov/boo
ks/NBK21603/ (Retrieved March 18 ,
2015)