The steps involved in the successful purification of intact RNA from cells, tissues and other samples are as follows:
- Disruption of Cells or Tissue
- Many mammalian cells require only minimal processing before RNA extraction.
- Muscle and connective tissue may require mincing or grinding.
- Rapid processing is important to protect RNA integrity.
- If immediate processing is not possible, process samples and store in a denaturing buffer as cold as possible before extracting RNA. Alternatively, freeze samples in liquid nitrogen before extraction.
- Inactivation of RNase Activity
- All methods of RNA isolation use strong denaturants to inhibit endogenous RNases and denature nucleoprotein complexes.
- There are two common kinds of denaturation methods used in RNA isolation: 4M guanidine isothiocyanate and phenol with SDS (sodium dodecyl sulphate).
- Removal of Contaminating DNA and Proteins
- Intact RNA is purified from contaminants by phenol:chloroform extraction.
- For further purification or concentration, RNA is often precipitated. Often for recovery of small quantities of RNA, a carrier such as RNase-free glycogen can be added.
Chief among these steps is the immediate inactivation of endogenous RNases, which are released from membrane-bound organelles upon cell disruption. RNA is notoriously susceptible to degradation, and special care is required for its isolation. All methods of RNA isolation use strong denaturants to inhibit endogenous RNases. RNases, in contrast with deoxyribonucleases (DNases), are difficult to inactivate because they do not require cofactors and are heat-stable. Some tissues such as pancreas and spleen are naturally rich in RNases, while other tissues such as liver are low in RNases. Ensuring that you maintain an RNase-free environment is critical to isolating intact RNA.