Molecular biomethods handbook

Abstract

The analysis of RNA is central to a wide range of molecular-biology studies, because It 1s often important to obtain informatlon about the expression of genes in hving organlsms Filter hybrldlzatlon of size-separated RNAs with a labeled nucleic-acid probe, a techinque known as northern-blot analysis, is designed to address the study of RNA sequences 1 This procedure, which derives from Southern-blot analysis (see Chapter 8 in this volume), is based on the ability of complementary single-stranded nucleic acids to form hybrid molecules 2 In brief, RNA is separated according to size on an agarose gel under denaturing condltlons, transferred (blotted) and lrreverslbly bound to a nylon or introcellulose membrane, and hybridized with a 32P-labeled nucleic-acid probe. After washing off the unbound and nonspecifically bound probe, hybridizing RNA molecules are revealed as autoradlographlc bands on a X-ray film 3,4