UNIT 10.11 Preparation of RNA from Tissues and Cells
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Ribaudo, R., Gilman, M., Kingston, R. E., Choczynski, P. and Sacchi, N. 2001. Preparation of RNA from Tissues and Cells. Current Protocols in Immunology. 3:V:10.11:10.11.1–10.11.14.
- Published Online: 1 MAY 2001
- Published Print: DEC 1992
Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA.
The first describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.