RNA Extraction from cells and tissues
RNA extraction is performed to purify the RNA from cell and tissue samples. Due to the presence of ribocholase enzymes, this method is very difficult and complex. Therefore, it is recommended that all equipment be rinsed with RNase neutralizing solutions, DEPC, and all steps are carried out under the hood with gloves.
There are several methods for RNA extracting, including :
- Phenol-chloroform method – 1
There are several methods for extracting RNA, and most common method is phenol-chloroform. This method is liquid-liquid extraction, which is used to purify nucleic acids and to remove proteins and lipids.
- Phenol-chloroform method – 2
In this method, an equal volume of phenol and chloroform is added to homogeneous tissues, lysed cells, and dissolved samples, then, the mixture is centrifuged, and two distinct phases of the water and organic are formed. Hydrophobic proteins and lipids are placed in the organic phase are on the bottom layer, and in the aqueous phase (upper) are the nucleic acids and other pollutants such as salt, sugar, and ….. When pipetting and separating the aqueous phase, care should be taken to avoid entering the organic phase, especially the upper part containing the protein, in the aqueous phase.
- Phenol-chloroform method – 3
The pH of the solution can play a determinative role To purify of RNA. In acidic conditions, DNA is transferred to the organic phase, and the RNA remains in the aqueous phase, and in the basic conditions, they will remain in the aqueous phase.
- Phenol-Chloroform Method – 4
Using isopropanol, the RNA is precipitated, and the last step is carried out with 70% -80% ethanol washing and purification. Finally, the precipitation of RNA is resolved in a given volume of RNase-free water.
The role of chloroform in RNA extraction :
Phenol solves about 10 to 15 percent of water and consequently reduced the RNA concentration. Additions of chloroform result in the release of water. The combination of chloroform with phenol intensifies the process of degradation of proteins and dissolves lipid.