In vitro 32P-labelling of viroid RNA for hybridization studies

Abstract

A method is described for the in vitro labelling of viroid RNA for use in hybridization studies. The citrus exocortis viroid (approximately 350 nucleotides) is degraded by hot formamide hydrolysis to fragments ranging from small oligonucleotides to near full lengths, and subsequently labelled to high specific activity by enzymatically attaching 32P to the 5'-end of each molecule. The cleavage step leaves 5' hydroxyl groups which allows the polynucleotide kinase to directly label the RNA fragments without prior enzymatic dephosphorylation. The method is simple, requires no special equipment, and provides a radioactive RNA probe sufficient for most types of hybridization studies.