This document discusses RNA isolation and its applications. It begins by describing the basic structure and functions of RNA and the three main types: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). It then explains that isolating and quantifying RNA is important to examine gene expression levels, splicing, and temporal and tissue-specific expression patterns. The document outlines the process of isolating RNA from tissues or cells, including breaking open cells and purifying the extracted RNA. It describes how to quantify RNA concentration using spectrophotometry and evaluates purity based on A260/A280 and A260/A230 ratios. In closing, some applications of isolated RNA are mentioned, including cloning genes, expression

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
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