An improved method for isolation of active vitellogenin messenger RNA from chicken liver. Use of diethylpyrocarbonate

Abstract

1. In chicken liver homogenate containing 15 mg protein and 1.2 mg RNA per ml, diethylpyrocarbonate (60 mM) at pH 6.5 and room temperature inactivated liver ribonucleases by over 90% whereas heparin (4 mg/ml) and yeast RNA (6 mg/ml) under similar conditions inhibited ribonuclease activity by 30–40%. 2. Maximal recovery in polysomes from homogenate containing 60 mM diethylpyrocarbonate required the presence of exogenous RNA. 3. The recovery in total mRNA and vitellogenin mRNA activity from liver homogenate can be improved by 3- and 7-fold, respectively, by adding 60 mM of diethylpyrocarbonate to the homogenization buffer containing already heparin and yeast RNA as inhibitors of ribonucleases. 4. Vitellogenin mRNA purified from homogenate containing diethylpyrocarbonate had a molecular weight of about 2.4 · 10 6 and coded for a major peptide of molecular weight 220 000 in a reticulocyte cell-free system.