IN-SITU ASSESSMENT OF MESSENGER-RNA ACCESSIBILITY IN HETEROGENEOUS TISSUE SAMPLES USING ELONGATION FACTOR-1-ALPHA (EF-1-ALPHA)

Elongation factor-1 alpha (EF-1 alpha) is an evolutionarily highly con served universal cofactor of protein synthesis in all living cells. In this study, its use as a positive control in situ hybridization assay s for specific detection of mRNA sequences was evaluated. Northern blo t analysis sis of Various non-neoplastic and neoplastic cultured cells of different stages of confluence, cell shape, and cell cycle status revealed that EF-1 alpha had a lower and more homogeneous expression t han did beta-actin. In situ hybridization assays using digoxigenin-lab eled riboprobes for the detection of EF-1 alpha mRNA in routinely form alin-fixed, paraffin-embedded tissue sections showed that EF-1 alpha i s a suitable positive control in all types of cells. However, variatio n of protease pretreatments demonstrated distinct and sometimes mutual ly exclusive digestion conditions for different cell types within the same tissue sample. Our results indicate that detection of EF-1 alpha mRNA is an appropriate internal standard for in situ hybridization ass ays and that it is useful to control artifacts such as false negatives caused by inappropriate protease pretreatments. The observed variabil ity of optimal protease pretreatments for different cell types within the same tissue section strengthens the importance of a positive contr ol in in situ hybridization assays.