The Isolation of Amoeba RNA of Low Protein Content in High Yields

Abstract

Comparison of six different RNA isolation procedures from amoebae showed that RNA recovery was increased by approximately 65% if pronase treatment was carried out prior to phenol extraction and was decreased by about 55%. and 17%. respectively if chloroform-isoamyl alcohol extraction or dialysis were carried out following phenol extraction. Dialysis was found to decrease contamination from protein or protein degradation products by 6–12 fold. Although pronase digestion appeared to increase protein contamination of the RNA preparation, the fact that chloroform-isoamyl alcohol extraction did not reduce it indicates that the contaminants were not protein. It is concluded that the most efficient scheme for isolating and purifying RNA from the free-living amoebae utilizes pronase digestion prior to phenol extraction and dialysis prior to RNA precipitation.