![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](https://image.slidesharecdn.com/rnaisolation-ss-190218123003/85/Isolation-of-RNA-and-its-application-8-320.jpg)
Isolation of RNA and its application
- 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
- 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