INTRACELLULAR LABELING OF BETA-ACTIN MESSENGER-RNA USING REVERSE-TRANSCRIPTASE INCORPORATED BIOTIN-DUTP INTO THE ACTIN CORTICAL MAT OF CORNEAL EPITHELIAL-CELLS

The intracellular distribution of bea-actin mRNA in whole embryonic co rneal epithelia is detected using primed in situ hybridization (PRINS) and analyzed with confocal laser microscopy. This is a relatively new technique that may be more specific in targeting mRNA faster than tra ditional in situ methods. The principle is that unlabeled oligonucleot ide primers complementary to specific mRNAs are used by the reverse tr anscriptase enzyme to synthesize cDNA in situ. The enzyme incorporates biotin-labeled dUTP into the complementary copy of mRNA. The intracel lular distribution of the biotin labeled mRNA copy is detected using a vidin-FITC combined with confocal microscopy. Confocal laser microscop y allows analysis of whole tissues in both the XY enface focal plane a nd the XZ cross-sectional focal plane. Human beta-actin primers show t hat the distribution of beta-actin mRNA is heavily concentrated at the optical plane that contains the actin-cortical mat. The label appears primarly in the basement-membrane zone (BMZ) of basal cells and with less intensity at the interface between the periderm and basal cells. These results differ from our previous beta-actin mRNA distribution st udies using traditional in situ hybridization techniques using the sam e oligonucleotide probe. In conclusion, this method may be useful for identifying areas of concentrated mRNA in tissues but appears to have a lower sensitivity than traditional in situ hybridization in whole ti ssue.