A NOVEL METHODOLOGICAL APPROACH TO STUDY THE LEVEL OF SPECIFIC PROTEIN AND GENE-EXPRESSION IN ABERRANT CRYPT FOCI PUTATIVE PRENEOPLASTIC COLONIC LESIONS BY WESTERN BLOTTING AND RT-PCR

Aberrant crypt foci (ACF) represent microscopic preneoplastic lesions, present in the carcinogen-treated rodent colons. The cellular and mol ecular changes occurring within these lesions may provide important cl ues to the sequence of events leading to advanced preneoplastic or neo plastic lesions. The main objective of this investigation was to deter mine whether intact mRNA and protein can be isolated from fixed tissue and studied. A pure population of ACF was harvested from colonic tiss ue that had been preserved in 70% ethanol or 10% buffered formalin, by using a dissecting microscope and plucking the ACF out using fine for ceps. The standard procedure of isolating RNA was performed successful ly on ACF and normal tissue preserved in 70% ethanol. The expression o f a housekeeping gene, beta-actin was demonstrated. Analysis of ethano l-preserved ACF for phosphorylated proteins was carried out by immunob lotting. The present study demonstrated that ACF are easily harvested from fixed tissues and that intact RNA and protein can be isolated fro m ACF or normal epithelium which can be used in techniques such as imm unoblotting and RT-PCR. Published by Elsevier Science Ireland Ltd.