Altered subcellular localization patterns of ferritin and beta-actin mRNAs in muscle cultures, resulting from incomplete penetration of digoxigenin-labelled riboprobes.

摘要: Protocols for in situ hybridization (ISH) of cultured cells often include storage alcohol at −20°C between fixation the cultures and ISH procedure. In experiments aimed localizing ferritin mRNA C2 muscle by with digoxigenin-labelled riboprobes, we have noticed that omission ethanol dramatically changed pattern localization. stored 50%, 70%, or 90% least 15 min, signal was stronger on myotubes than myoblasts but uniformly distributed over both. untreated cultures, patchy, concentrated extremities elongated very sparse myotubes. Similar results were obtained a probe to β-actin used as control, except higher all conditions. When probes reduced size <100 bases from 561 1150 actin, became uniform, regardless prehybridization treatment. The patchy disappeared when treated RNase A following hybridization, suggesting it is non-specific, despite its absence hybridized sense probe. We conclude incomplete access RNA can result not only also artefactual patterns