A novel in situ double-labeling method for simultaneous detection of mRNA and expressed protein or two different...

We have developed a novel double-labeling method to investigate multiple gene expression in single cells. The method relies on the use of a radioactive probe followed by a colorimetric probe. Unique to this method, the radioactive signal is first captured on an emulsion pre-coated slide, which totally separates it from the process of color development and prevents any interference with the radiolabeled probe. We cite two applications of the new procedure: (1) to study the correlation between acetylcholine receptor (AChR) alpha-subunit mRNA and protein expression in cultured chick myoblasts and (2) to investigate the co-expression of (AChR) alpha and gamma mRNAs in nascent myotubes. In the former case, the radioactive signal is generated by incubation of live cells with 125I-alpha-bungarotoxin, in the latter, by an in situ hybridization (ISH) with 35S-labeled DNA probe. Colorimetric labeling is accomplished in a second step by ISH using digoxigenin-labeled oligos. Analysis of 203 myoblasts showed that AChR alpha-subunit protein and mRNA are co-expressed. Examination of 4-day-old myotubes suggested that most, but not all, nuclear clusters co-express alpha and gamma mRNAs. These results demonstrate that the described protocol has high sensitivity and specificity for detection of protein and message, or two different mRNAs on a single cell level.