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 molecular changes occurring within these lesions may provide important clues to the sequence of events leading to advanced preneoplastic or neoplastic lesions. The main objective of this investigation was to determine whether intact mRNA and protein can be isolated from fixed tissue and studied. A pure population of ACF was harvested from colonic tissue that had been preserved in 70% ethanol or 10% buffered formalin, by using a dissecting microscope and plucking the ACF out using fine forceps. The standard procedure of isolating RNA was performed successfully on ACF and normal tissue preserved in 70% ethanol. The expression of a housekeeping gene, beta-actin was demonstrated. Analysis of ethanol-preserved ACF for phosphorylated proteins was carried out by immunoblotting. The present study demonstrated that ACF are easily harvested from fixed tissues and that intact RNA and protein can be isolated from ACF or normal epithelium which can be used in techniques such as immunoblotting and RT-PCR.
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Department of Foods and Nutrition, University of Manitoba, Winnipeg, Canada. bird@bldghumec.unmitoba.lani.ca
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D000818:Animals; D001706:Biopsy / Q000379:methods*; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D007413:Intestinal Mucosa / Q000378:metabolism / Q000473:pathology; D008297:Male; D016133:Polymerase Chain Reaction; D011230:Precancerous Conditions / Q000378:metabolism* / Q000473:pathology; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344
D011506:Proteins; D012333:RNA, Messenger