Non-isotopic in situ hybridization of CD44 transcript in formalin-fixed paraffin-embedded sections

We established a protocol for the non-isotopic in situ detection of adhesion molecule CD44 messenger RNA (mRNA) in archival formalin-fixed paraffin-embedded sections of human surgical materials. Four brain tumor samples with different histopathologies (a metastatic adenocarcinoma, a metastatic squamous carcinoma, a glioblastoma and a craniopharyngioma) were thus studied using a 157 nt digoxigenin-labeled RNA probe complementary to the common mRNA region to all the CD44 isoforms. The CD44 transcript was detected in the cytoplasm of glioma and such epithelial tumor cells as metastatic carcinoma and craniopharyngioma. A competitive hybridization study confirmed the specificity of the CD44 probe. The optimization of critical conditions are also discussed. This protocol should therefore be useful in making an accurate evaluation of mRNA localization and may also facilitate the successful completion of extensive retrospective studies on a large number of archival samples.