Lecturer Bahiya Osrah

 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 STRUCTURE OF RNA

RNA TYPES

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

SIRNA (Gene Silencing) Degrades mRNA then it inactivates the gene translation

MIRNAS Tiny 21–24-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 is eukaryotic microorganisms that belong to the kingdom of fungi; there are 100,000 species or more. This experiment Saccharomyces cerevisiae (Baker’s 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. YEAST

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 REAGENTS AND USES

1.Suspend 5 g of dried yeast in 30ml of water previously heated to 37°C 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. RNA + polysaccharides DNA + Protein 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 PROTOCOL

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 RNA Protein

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. PROTOCOL

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 HOW GOOD OUR ISOLATION??

Compare your product with a commercial preparation by measuring the pentose, phosphorus, and DNA content and determining the absorption spectrum. Keep your preparation for use in later experiments.