title: Comparison of isotopic and non-isotopic labelling for in situ hybridisation of various mRNA targets with cRNA probes.
abstract: In situ hybridisation methods to localise messenger ribonucleic acid (mRNA) targets in tissue sections or cell preparations using riboprobes can be successful with either isotopic or non-isotopic labelling. Investigators often wish to decide which labelling method provides the maximum specificity, sensitivity and resolution, with minimum nonspecific background. In this study we compared isotopic (35S) and non-isotopic (digoxigenin) labelling, using a variety of probes and paraffin-embedded tissues. The targets were human beta-actin and von Willebrand Factor mRNAs in archival human tissues; and mRNAs for two closely related trefoil factor family (TFF) peptides, TFF2 and TFF3, in rat duodenum. Patterns of localisation with both isotopic and non-isotopic probes were broadly similar for each target. The 35S labelling provided good contrast and sensitive detection under darkfield illumination, but the cellular or subcellular resolution of the target was less precise than that obtained with the digoxigenin-labelled probes in transmitted light. Digoxigenin labelling in individual cells was more clearly demonstrated, but occasionally the contrast of positive staining with background was poor. The sensitivity of each method appeared to be similar for these high-abundance targets, therefore the choice between isotopic and non-isotopic labels is dependent upon the aim of the study and the cellular resolution required.
authors.affiliation: Histopathology Unit, Imperial Cancer Research Fund, London, UK.
authors.forename: J H
authors.initials: JH
authors.lastname: Steel
authors.forename: R E
authors.initials: RE
authors.lastname: Jeffery
authors.forename: J M
authors.initials: JM
authors.lastname: Longcroft
authors.forename: L A
authors.initials: LA
authors.lastname: Rogers
authors.forename: R
authors.initials: R
authors.lastname: Poulsom
mesh_terms: D000818:Animals; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007553:Isotope Labeling; D015347:RNA Probes; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D000071164:Trefoil Factor-2
chemical_list: D015347:RNA Probes; D018075:RNA, Complementary; D012333:RNA, Messenger; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2