Formal Rep 1

ISOLATION AND CHARACTERIZATION OF RIBONUCLEIC ACID (RNA)
Group 2-2KMT*Camenforte, L.L., Bustos, M.V.N., Cabansag J.M., Calero J.L., Cardona, N.D.,Ching,S.J.
ABSTRACT
The isolation of ribonucleic acid (RNA) from yeast (Saccharomyces cerevisiae) was done by
extracting the nucleic acids and soluble proteins through heating the sample with sodium
hydroxide. To test the purity after hydrolysis, chemical characterization test was performed to
both standard solutions, for control, and to be hydrolyzed RNA solution. Upon conclusion of
the experiment, the test performed on the hydrolyzed RNA solution yielded a negative result
when compared to the results given out by the standard solutions. The test for purines gave
out a yellow solution for adenine and a red layered solution for guanine. The test for
pyrimidines result in a purple solution, red to blue litmus paper for cytosine and uracil.
INTRODUCTION
Ribonucleic acid, or RNA is one of the three This leads to formation of the so-called
major biological macromolecules that are "sugar-phosphate backbone", from which
essential for all known forms of life (along the nitrogenous bases project. It is also one
with DNA and proteins). Ribonucleic acid of the three major macromolecules that
(RNA) molecules are single-stranded are essential for all known forms of life. The
polymer nucleic acids made up of three major types of RNA in eukaryotes
ribonucleotides. Each ribonucleotide are involved in essential process of protein
consists of a ribose sugar, a phosphate synthesis. The mRNA carries the
group, and a nitrogenous base derived from information from the DNA in the nucleus to
purines which are adenine and guanine, the site of protein synthesis, tRNA
and from pyrimidines, namely, cytosine and delivers amino acids to the ribosomes and
uracil. Unlike DNA, RNA is relatively shorter rRNA links amino acids together finally
which makes it less susceptible to shearing; forming the proteins.
however, the same is also the cause for its
weaker stability making it more prone to
degradation.
Figure 1.2 Process of protein

synthesis
Figure 1.1 Structure of a Yeast (Saccharomyces cerevisiae) are
Ribonucleotide (adenosine 5`- single-celled microorganisms that are
monophosphate) classified, along with molds and
mushrooms, as members of the Kingdom
Fungi. Yeasts are evolutionally diverse and
Added to this RNA is synthesized in the are therefore classified into two separate
cell by enzymes, RNA polymerase, and phyla, Ascomycota or sac fungi and
involves forming phosphodiester bonds Basidiomycota or higher fungi that together
between the 3' carbon of one nucleotide form the subkingdom Dacrya.
and the 5' carbon of another nucleotide.
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Although yeast is single-celled organisms,  Concentrated sulfuric acid
they possess a cellular organization similar
to that of higher organisms, including  Barium hydroxide
humans. Specifically, their genetic content  10% (NH4)2MoO4 solution
is contained within a nucleus. This classifies
them as eukaryotic organisms, unlike their  Br2 water
single-celled counterparts, bacteria, which
do not have a nucleus and are considered  Orcinol reagent
prokaryotes.  UV-Vis spectrophotometer
 Quartz cuvettes
 Marbles
Standard solutions used:
 Ribose,adenine,guanine,
cytosine, and uracil
Other materials used:
 Active dry yeast, 1% sodium
hydroxide, glacial acetic acid,
concentrated hydrochloric
acid, 95% ethanol, ether,
litmus paper, cheesecloth
Figure 1.3 2D diagram of yeast cell
B. Procedure
Bial’s Test (or more commonly known as RNA Isolation from yeasts
“Test for Ribose”) is a confirmatory test
used to distinguish pentose from hexose. In a beaker, 5.0 mL of the 1% sodium
The test would produce a blue-green hydroxide solution was diluted with 25 mL
colored solution if a pentose is present in of water and added to 3.0 g of active dry
the solution. yeast. The solution was heated in a 60 C
water bath for 15 minutes and stirred
Murexide Test is a confirmatory test used occasionally. The solution was strained
to detect the presence of purines and through a cheesecloth and centrifuge for 2
purine derivatives. The test will produce a minutes and a drop wise glacial acetic acid
red residue if a purine is present. was added to the cooled supernatant until
Wheeler-Johnson’s Test is a confirmatory it was faintly acidic to the litmus.
test used to detect the presence of
pyrimidines which will produce a purple The supernatant was evaporated up to
solution if it is present in a given solution. approximately 10mL over a water bath. The
supernatant was again centrifuge and
The Test for Phosphate is done on a filtered and was cool to a room temperature
solution to confirm the presence of a 40 C or lower. In a large test tube the
phosphate group in a given solution. The solution was pour into a 20 mL of the 95%
test will produce a yellow precipitate if the ethyl alcohol containing 0.2 mL of the
solution is positive of a phosphate group. concentrated Hydrochloric acid while
vigorously stirring it. The RNA was settled
in a tall vessel and covered with a paraffin
EXPERIMENTAL wax and refrigerated until the next
laboratory period. The RNA was pour and
A. Compound tested (or sample used) washed twice with 5 mL of the 95% ethanol
and twice with ether.
Reagents used:
Ultraviolet Measurement of Isolated
 0.3 M sodium hydroxide
RNA
 10% potassium hydroxide
The aliquot was diluted with more TE buffer  One mL of water was then added
(0.5 ml of the aliquot with 4.5 ml of the to the colorless liquid and was
buffer). The solution was then transfer to a
heated in a boiling water bath for 5
quartz cuvette and the absorbance was
determined at 260nm, 280 nm, and 230 minutes.
nm.  After heating, the mixture was
cooled. To the cooled mixture, 1
CHEMICAL CHARACTERIZATION mL of 10% (NH¬4)2MoO4 solution
1. Alkaline Hydrolysis was added.
 To a small amount of RNA isolate,  The mixture was mixed well and
2 mL of 0.3 M NaOH was added was diluted with 10 mL of water.
 The mixture was then heated in a  Lastly, the diluted mixture was
boiling water bath for an hour. allowed to stand for 5 minutes.
 The hydrolysate was then cooled The color of the precipitate formed
and adjusted to ph 4-6 using was taken note of.
glacial acetic acid.
4. Test for Purines (Murexide
Test)
2. Test for Ribose  In two evaporating dishes, 5-
 Two mL of orcinol reagent was 10 drops of the nucleic acid
added to 0.5 mL of hydrolyzed RNA solution and the standard
solution adenine/guanine solution
 The mixture was then placed in a respectively were added.
water bath for 5 – 10 minutes.  The solutions were carefully
 The same procedure was repeated evaporated until dry in a
water bath.
replacing the hydrolyzed RNA with  The residues formed were
the standard ribose solution used then moistened with 10% KOH
as a control. and were further heated.
 The color of the solution of both  The color changes for both
the RNA and the standard were the addition of KOH and the
taken note of. subsequent heating were
taken note of.
 After heating, a few drops of
3. Test for Phosphate
water was added to both, the
 Two test tubes were prepared, one solution’s color was taken
containing 1 mL of nucleic acid note of as well.
solution and another containing 1  The solutions were
mL of standard phosphate solution evaporated once more and
 To each tube, 1 mL of conc. H2SO4 the colors of the residues
was added. were taken note of.
 Both tubes were heated over a 5. Test for Pyrimidines
small flame and were shaken (Wheeler-Johnson Test)
frequently until the contents  0.5 mL of nucleic acid solution
turned brown. and 0.5 mL of standard
 Upon turning brown, the mixture cytosine/uracil solution, in
was cooled. separate tubes, were treated
with an excess of bromine
 To the cooled mixture, 0.5 mL
water until the solution turned
conc. HNO3 was added. The yellow.
mixture was then heated again  Excess was removed by
until white fumes appeared. boiling both tubes until it
 The addition of conc. HNO¬3 and turned light yellow or
the heating were repeated until colorless
the liquid was colorless.  Excess Ba(OH)¬2 was then
added to both and were
This study source was downloaded by 100000857575723 from CourseHero.com on 11-27-2022 19:27:12 GMT tested
-06:00 with litmus paper.
 The solutions’ colors were inferred as the solution not having a purine
taken note of. base.
RESULTS AND DISCUSSION
Table 2.1 Chemical Characterization
Chemical Test RNA from Yeast

Adenine; Yellow
Test for Purines solution
Guanine; Red layer
solution
Test for Cytosine and Uracil;
pyrimidine purple solution, red to
blue litmus paper
As seen in the table above, the following Figure 3.2 Test for pyrimidine
tests yielded positive results for the (Cytosine and Uracil)
standard solutions. The presences of a
phosphate group are confirmed when the
formation of a yellow precipitate is Lastly, the Wheeler-Johnson Test or more
observed due to the hydrolysis of commonly known as the Test for
pyrophosphate to phosphate. The Pyrimidines produce a purple solution when
hydrolyzed RNA solution did not produce there is a pyrimidine base in the solution
any precipitate which can be interfered as due to the formation of 5, 5-dibromo-6-
the solution not having a phosphate group. hydroxyhydro derivatives. A white turbid
solution, upon performing the test, was
produced confirming the absence of a
pyrimidine base in the hydrolyzed RNA
solution.
Figure 3.1 Synthesized Ribonucleic

Acid
A characteristic red residue is observed

when the test for purines is performed. This
residue is due to the degradation of the
purine and the formation of murexide. The
hydrolyzed RNA solution produced a
brownish-yellow solution which can be Figure 3.3 Test for pyrimidine
(red to blue litmus paper)
method for the isolation of RNA from
plant tissues. Analytical Biochemistry
163(1), 16-20.
REFERENCE
[6] Walker, J. M., & Rapley R (2000).
Molecular biology and biotechnology
[1] Baker, S. S., Rugh, C.L. & (4th ed.). Cambridge, UK: Royal
Kamala, J.C. (1990). RNA and DNA Society of Chemistry
isolation from recalcitrant plant tissues.
BioTechniques 9(3), 268-272.
[7] Campbell, M.K., Farell, S.O.
[2] Bernas, G. C., Ysrael, M. C., & (2009). Biochemistry. 6th ed.
Bernaldez, A.T. (1994). Basic Philippines: Cengage Learning Asia
laboratory studies in biochemistry Pte. Ltd.
(3rd ed.). Manila: UST Publishing
House. [8] Crisostomo, A., et.al. (2010).
Laboratory manual in general
[3] Campbell,M.K., & Farrel, S.O. biochemistry. Quezon City,
(2006). Biochemistry (5th ed.) Philippines: C&E Publishing Inc.
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[4] Concepcion, G.P., de Guzman, T.S. [9] Liu, C.M. (n.d.). Arabidopis RNA
& Ticson, Ma. V.N. (Eds). (1998). extraction protocol. Retrieved from
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techniques in experimental bidopsisrna.html
biochemistry: laboratory guide (2nd
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Philippines. /949.html
[5] Longemann, J., Shell, J., &

Willmitzer, L. (1987). Improved
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ISOLATION AND CHARACTERIZATION OF RIBONUCLEIC ACID (RNA)

Figure 1.2 Process of protein

RESULTS AND DISCUSSION

Table 2.1 Chemical Characterization

Chemical Test RNA from Yeast

Figure 3.1 Synthesized Ribonucleic

A characteristic red residue is observed

[5] Longemann, J., Shell, J., &

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