UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY
MANILA, PHILIPPINES

RNA EXTRACTION AND DETECTION FROM YEAST

Manalo, P.R., Quibol, S. M., Raagas, E., Rosario, C.F., Tabula, M.C., Tan, J.M., Tornea, E.M.

ABSTRACT
Ribonucleic acid is one of the three major biological macromolecules that are essential for all known
forms of life molecules, consisting of a single-stranded chain of phosphate and ribose units with a
nitrogenous base derived from purines. The objectives of the experiment are to isolate RNA from yeast,
determine its purity through UV spectroscopy, and characterize RNA after basic hydrolysis. The RNA
molecules were obtained from RNA isolation from yeast and was divided into portions of 20 uL for UV
quantification, with the remaining portion for hydrolysis. The obtained product from the isolation
process yielded an RNA isolate, which appeared to be a powdery sediment, dirty white in color. It
underwent the UV analysis, where the absorbance at 230 nm, 260 nm, and 280 nm readings were
obtained and used for further specifications. The RNA purity was further assessed using the A260/A280
and A260/A230 ratio. The experiment yielded quantitative results lower than the required amount of 1.8
to 2.0 for a good quality isolated RNA sample, and this signifies a contamination with protein. The
isolated RNA failed to meet the standard of a pure isolation.

INTRODUCTION The isolation of RNA molecules varies

Ribonucleic acid (RNA) molecule is one of the
three major biological macromolecules that are
essential for all known forms of life. It consists
of single-stranded chain of phosphate and
ribose units with a nitrogenous base derived
from purines which are guanine (G) and adenine
(A) and from pyrimidines which are cytosine (C),
uracil (U), and thymine (T). RNA is used in all
steps of protein synthesis and carries the
genetic code for most viruses. In comparison
with DNA, RNA molecules are relatively shorter,
making it more susceptible to shearing and
degradation by ribonucleases.
according to the type of tissue employed and
the RNA species to be isolated. The RNA sample
was isolated from yeast (Saccharomyces
cerevisiae), a unicellular fungus that contains
4% RNA by weight. It directly involves heating
with alkali (NaOH) which loosens and lyses the
cell membrane leading to the extraction of RNA.
Increased pH level by adding glacial acetic
inactivates nucleases resulting to the prevention
of RNA degradation. Furthermore, in order to
get rid of contaminants such as lipids and
unnecessary supernatant, the sample RNA
needs to be filtered and washed with ethanol
containing HCl then centrifuged.

The most widely used traditional method for

assessing RNA concentration and purity is by UV
spectroscopy. The absorbance of a diluted RNA
sample is measured at 260 and 280 nm, with
the nucleic acid concentration to be calculated
using the Beer-Lambert law, which predicts a
linear change in absorbance with concentration.

Figure 1. Ribonucleotide Components


Figure 2. Beer-Lambert’s Equation
Using this equation, an A260 reading of 1.0 is
equivalent to approximately 40 µg/ml single-
stranded RNA. The A260/A280 ratio is used to
assess RNA purity, a ratio ranging from 1.8-2.1
is indicative of highly purified RNA. In order to
separate its components for specific tests,
hydrolysis of the nucleic acid is done by adding
NaOH to the remaining RNA sample solution
followed by the addition of glacial acetic acid to
adjust its pH to 4-6.
The objectives of this experiment were to
isolate RNA from yeast, to determine its purity
by UV spectroscopy and to characterize it after
basic hydrolysis.
METHODOLOGY
The materials used for the isolation of yeast
included an active dry yeast (Saccharomyces
cerevisiae), concentrated HCl, glacial acetic
acid, 1% NaOH, and 95% ethanol.
RNA Isolation from Yeast
The RNA sample was obtained by
precipitating and isolating the RNA from yeast
(Saccharomyces cerevisiae) using 5.0 mL of the
1% NaOH solution with 25 mL water. The
solution was heated in a water bath at 60°C for
15 minutes, then strained in a cheesecloth with
the filtrate centrifuged afterwards. Glacial acetic
acid was added to the supernatant liquid until it
was faintly acidic. The solution was evaporated
until it was reduced to 10 mL and was cooled
below 40°C. The recovered solution was then
poured into a 20mL 95% ethyl alcohol
containing 0.2 mL of HCl and was stirred
vigorously. The RNA was allowed to settle at the
bottom of the centrifuge tube.
Preparation of RNA Sample
The isolated RNA sample was diluted with
2mL of the Tris-EDTA buffer, forming a solution.
It was then divided into two portions — (a) 20
uL portion for UV quantification with the (b)
remaining solution for the hydrolysis of RNA.
A. UV Analysis
In the 20 uL RNA sample solution, a 980 uL
of Tris-EDTA buffer was added and provided a
total volume of 10 mL. This was read in UV
spectrometer at 230 nm, 260 nm, and 280 nm
respectively. The ratio of A260/A280 was
calculated and compared to A260/A230 for the
assessment of the purity of the RNA isolate. The
total RNA was computed using the formula
below:
Total RNA(μg) = A260 x dilution factor x 40 x
sample volume
B. RNA Hydrolysis
For the RNA hydrolysis, 5 mL of 0.3 NaOH
was added to a test tube containing the
remaining solution of RNA. The solution was
then incubated in a water bath for 60 minutes.
It was cooled afterwards and acidified to pH 4-
6 by glacial acetic acid and was transferred to
the nucleic acid hydrolysates in a
microcentrifuge tube.
RESULTS AND DISCUSSION
The RNA was extracted by heating with NaOH
and separated from associated proteins through
acid extraction at pH 4.5. It was then treated
with alcohol and ether to eliminate its lipid
components. The obtained product of the
isolation process yielded an RNA appearing as a
powdery sediment which is dirty white in color.
Ultraviolet analysis was performed, and the
absorbance of the sample was determined at
230 nm, 260 nm, and 280 nm. The results are
shown in the table below.
Table 1. Ultraviolet Measurement of
Isolated RNA from Yeast
UV MEASUREMENT
RNA
230 nm 260 nm 280 nm
Yeast 1.5436 1.8604 1.5277
As seen in Table 1, the highest absorbance
was recorded at 260 nm which is the ^max for
nucleic acids. Presence of exposed nitrogenous
bases was therefore detected resulting to
hyperchromic effect. This effect caused the
absorbance to be higher compared to the
absorbance at 230 nm and 280 nm.
Furthermore, ^max for protein contaminants
was at 280 nm.
CONCLUSION
Upon the addition of NaOH for isolation and
treatment of alcohol to eliminate the lipid
content, it yielded a powdery, dirty white
sediment. The isolated RNA presented a positive
protein concentration based on the A260/A230
ratio, depicting a negative result for a successful
pure isolation.
REFERENCES
 What is RNA? (n.d.). Retrieved from
https://www.rnasociety.org/about/what-is-
rna/.\

Using the formula, A260 x dilution factor

x 40 x sample volume, the total RNA was
computed to be 1488.32. The RNA purity was
further assessed using the A260/A280 and
A260/A230 ratio.

Table 2. Total RNA Computation

UV Measurement

(A260/A280) (A260/A230)

1.8604/1.5277 = 1.8604/1.5436 =
1.2178 1.2052

A pure isolated RNA sample of good quality

ranges from 1.8-2.0 ratio for A260/A280. A higher
value signifies phenol contamination while a
lower value means there is a protein
contamination. Thus, the RNA isolated from
yeast in the performed experiment, based from
the resulting A260/A280 ratio, shows that there is
a protein contamination, with the values lower
than the desired amount of a good quality RNA
sample.

Furthermore, A260/A230 ratio of 2.0 signifies

presence of other contaminants such as
polysaccharides and salts as these contaminants
are absorbed strongly at 230 nm. Therefore, the
presence of these contaminants in the sample
was not observed since the ratio did not meet
the ideal ratio of 2.0.