Molecular‐Weight Determination of Animal‐Cell RNA by Electrophoresis in Formamide under Fully Denaturing Conditions on Exponential Polyacrylamide Gels

Abstract

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA · RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45° and 55 °C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10⁴–10⁷. Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights, the size of animal cell RNA was reexamined. Precursor rRNA (45 S) from HeLa cells migrates according to a molecular weight of 4.1 × 10⁶. Nascent precursor mRNA has molecular weights of up to 5 × 10⁶ in the case of duck erythroblasts and of up to 10⁷ in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.