INTRACELLULAR LABELING OF BETA-ACTIN MESSENGER-RNA USING REVERSE-TRANSCRIPTASE INCORPORATED BIOTIN-DUTP INTO THE ACTIN CORTICAL MAT OF CORNEAL EPITHELIAL-CELLS
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abstract
The intracellular distribution of -actin mRNA in whole embryonic corneal 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 traditional in situ methods. The principle is that unlabeled oligonucleotide primers complementary to specific mRNAs are used by the reverse transcriptase enzyme to synthesize cDNA in situ. The enzyme incorporates biotin-labeled dUTP into the complementary copy of mRNA. The intracellular distribution of the biotin labeled mRNA copy is detected using avidin-FITC combined with confocal microscopy. Confocal laser microscopy allows analysis of whole tissues in both the XY en face focal plane and the XZ cross-sectional focal plane. Human -actin primers show that the distribution of -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 -actin mRNA distribution studies using traditional in situ hybridization techniques using the same 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 tissue. 1994.
