rna isolation

1. RNA Isolation and Technology
Applications
By
S.Selvajeyanthi
Assistant Professor
Dept of Microbiology
Tiruppur kumaran college for women
Tirupur

2. Nucleic Acid Chemistry
• DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid)
store and transfer genetic information in living
organisms.
• RNA:
– found in the nucleus and the cytoplasm
– key to information flow within a cell

3. Structure of RNA nucleotides

4. The Central Dogma

5. Cellular “total” RNA
• Messenger RNA (mRNA): 1-5% Serves as a
template for protein synthesis
• Ribosomal RNA (rRNA): >80% Structural
component of ribosomes
• Transfer RNA (tRNA): 10-15% Translates
mRNA information into the appropriate amino
acid

6. Why Isolate RNA?
Messenger RNA synthesis is a dynamic expression
of the genome of an organism. As such, mRNA is
central to information flow within a cell.
• Size – examine differential splicing
• Sequence – predict protein product
• Abundance – measure expression levels
• Dynamics of expression – temporal,
developmental, tissue specificity

7. RNA isolation
Tissue Cells
Break open the cell
RNA Purification
Quality
Parameters
Purity
Integrity

8. Quantitation of RNA
• Nucleic acids absorb UV light maximally at
260nm
• For RNA:
1 OD260 Unit = 40 μg/ml of ssRNA
• The concentration in the sample is
calculated
by using the formula:
A260 x dilution x 40 = [RNA] μg/ml

9. Estimating RNA purity by
spectrophotometry
• A260/A280
– pure RNA will exhibit an A260/A280 ratio within the range
of 1.8 - 2.0
– If the RNA exhibits a ratio lower than 1.7, this indicates
protein contamination in your sample
• A260/A230
– properly purified RNA should exhibit an A260/A230 ratio
within the range of 1.8 - 2.0.
– If the RNA exhibits a ratio lower than 1.7, this indicates
guanidine contamination in your sample
• While spec readings do give an estimation of purity, they do
NOT indicate RNA integrity

10. Applications
• Clone genes
• Expression Studies
• Northern blotting
• cDNA synthesis and gene product evaluation
• Microarray
Thank U