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

Protocols for in situ hybridization (ISH) of cultured cells often include s torage in alcohol at -20 degrees C between fixation of the cultures and the ISH procedure. In experiments aimed at localizing ferritin mRNA in C2 musc le cultures by ISH with digoxigenin-labelled riboprobes, we have noticed th at omission of the ethanol storage dramatically changed the pattern of mRNA localization. In cultures stored in 50%, 70%, or 90% ethanol for at least 15 min, ferritin signal was stronger on myotubes than myoblasts bur. was un iformly distributed over both. In untreated cultures, the signal was patchy , concentrated on the extremities of the elongated myoblasts and very spars e in myotubes. Similar results were obtained with a probe to p-actin used a s a control, except that signal was higher in myoblasts in all conditions. When the probes were reduced in size to similar to 100 bases from 561 for f erritin and 1150 for actin, the pattern became uniform, regardless of prehy bridization treatment. The patchy pattern disappeared when cells were treat ed with RNase A following hybridization, suggesting that it is non-specific , despite its absence in cultures hybridized with a sense probe. We conclud e that incomplete access of RNA probes can result not only in a reduced ISH signal but also in artefactual patterns of mRNA localization.