Extraction and fractionation of low molecular weight RNA on the microscale.

Abstract

SEPARATION techniques involving gel electrophoresis recently introduced for high molecular weight RNA molecules are most useful, compared with sedimentation analysis, in making possible increased resolution of migrating RNA species. Electrophoretic techniques are also valuable because they can be scaled down to make analysis of RNA from the single cell relatively easy. Single cell analysis was originally developed by Daneholt et al.1, who used agarose gels for separating the ribosomal RNAs and 4S RNA. This purpose, however, can be served better with the use of composite polyacrylamide–agarose gels that improve separation (see following communication). Separation of the relatively low molecular weight species of RNA (species smaller than 16S size) on a small scale is, on the other hand, best carried out in relatively concentrated polyacrylamide gels. We describe here the isolation and separation of RNA from individually isolated cell components from salivary gland polytene cells. We also show that the information which can be obtained in this way is of unique value, because well defined structural components can be isolated separately from a fixed tissue preparation without exposing the tissue components to aqueous fluids during the isolations, with the consequent uncertainties about the origin and native state of the analysed nucleic acid.