Northern Blotting of RNA Denatured in Glyoxal Without Buffer Recirculation

Abstract

A rapid and easy procedure for preparing Northern blots is described. RNA is denatured with glyoxal in the presence of ethidium bromide and glycerol, then electrophoresed through agarose in a buffer that does not require recirculation. Without any additional washes, the RNA is vacuumblotted to a nylon membrane in NaOH, which simultaneously removes the glyoxal adducts. All of these steps plus prehybridization of the filter and addition of a digoxygenin-labeled probe can be completed in one day. Using standard procedures to wash the filters and detect the probe, the entire procedure can be completed within two days.