RNA

ISOLATION
LAB 3
Lecturer
Bahiya Osrah
 STRUCTURE OF RNA

RNA exist as a single strand.

Ribose Sugar (5 carbon sugar)
Phosphate group
Adenine, Uracil, Cytosine, Guanine
For RNA, nucleosides are formed similarly to DNA.
Hairpin is a common secondary/tertiary structure.
Double stranded RNA can also exists in mammals
RNA TYPES
 NEW TYPES OF RNA

snRNA - Small nuclear RNA

is a class of small RNA molecules that are found within the nucleus of
eukaryotic cells.
They are involved in a variety of important processes such as RNA
splicing (removal of introns), regulation of transcription factors and
maintaining the telomeres.
snoRNA - Small nucleolar RNA
snoRNAs are involved in rRNA modification.
They are located in the nucleolus .
Other RNAs involved in Gene Expression Regulation:
siRNA- RNA interference
miRNAs- Micro RNA
 SIRNA

(Gene Silencing)
Degrades mRNA then
it inactivates the gene
translation
 MIRNAS
Tiny 2124-nucleotide RNAs
Non coding small RNAs
unlike siRNAs, miRNAs downregulate expression
after translation initiation without affecting mRNA
stability
stem-loop structure is highly Conserved

Translation
Inhibition
 YEAST

Yeast is eukaryotic microorganisms that belong to the kingdom of

fungi; there are 100,000 species or more.
This experiment Saccharomyces cerevisiae (Bakers yeast) is used in
baking.
It is one of the most studied eukaryotic model.
It reproduces by division process known as budding.
Many proteins in human biology were first discovered by studying
their homologs in yeast. S. cerevisiae was the first eukaryotic genome
to be completely sequenced.
The genome composed of 13,000,000 base pairs, 6275 genes only
5,800 genes are functional.
It was estimated that yeast shares 23% of its genome with humans.
 REAGENTS AND USES

Total yeast RNA is obtained by extracting a whole cell homogenate with

phenol.
The concentrated solution of phenol:
Disrupts hydrogen bonding in the macromolecules, causing denaturation of
the protein.
Alcohol and K-acetate:
To precipitate RNA.
The product obtained is free of DNA but usually contaminated with
polysaccharide.
Amylase:
To degrade polysaccarides
 RNA +
polysaccharides

PROTOCOL
DNA + Protein

1. Suspend 5 g of dried yeast in 30ml of water previously heated to 37C in

beaker. Leave it for 15 min at this temperature.
2. Add 25 ml of concentrated phenol solution in the hood (Care:
corrosive)(protein denaturation).
3. Stir the suspension by using a stirring magnet for 30 min at room
temperature.
4. Centrifuge at 5000 rpm for 7 min.
5. Collect the upper aqueous layer with a Pasteur pipette
(Upper layer contains RNA and polysaccharides).
6. Centrifuge at 5000 rpm for 7 min to sediment denatured protein.

7. Collect the upper layer in measuring cylinder and measure its volume.
 PROTOCOL

8. Add potassium acetate to the supernatant to a final

concentration of 20 g/L (Note: every 9 ml of supernatant
add 1 ml of potassium acetate)

9ml 1ml K-acetate

X ml Y? ml K-acetate
Y= (1) (x)
9
 Protein

PROTOCOL
RNA

9. Precipitate the RNA by adding 2 volumes of ethanol means Add

ethanol 2( X+Y).
10. Cool the solution in ice and leave to stand for 1 h.
11. Centrifuging at 2000 rpm for 5min in the cold.
12. Collect the precipitate= remove the supernatant.
13. Record the wt of empty filter paper (w1).
14. Wash the RNA with 1ml of ethanol-water (3:1) depend on the
amount of precipitate.
15. Filter the solution and then add 3 ml of ethanol to the filter paper.
16. Finally, wash with 3 ml ether
17. air dry, and weight.
 PROTOCOL

X= filter paper wt
Y= RNA + Filter paper
Z= Y-X (RNA WT only)
YEILD % = Z (100)
5
10. Dissolve RNA powder in 10 ml, 1% NaOH.
 HOW GOOD OUR ISOLATION??

Yeast contains about 4 per cent RNA by dry weight.

100 g of yeast contains 4g RNA so.
100 g yeast 4g RNA
5g yeast x g RNA
X= 0.2% or 0.2 g
Then compare this X with your yield
YIELD % = Z (100)
5