A method to detect the expression of genes biorreguladores in microculturas used eucarioticas

摘要

A method allowing accurate and sensitive detection of a transient expression of bioregulatory genes in microcultures of eukaryotic cells. The method comprises the steps of (a) isolating and culturing the cells under conditions inducing the expression of at least one of the said genes, (b) lysing the cells with guanidinium hydrochloride, (c) selectively precipitating ribonucleic acid by adding to the homogenised cell preparation a sodium or potassium acetate solution followed by absolute ethanol, (d) recovery and blotting RNA onto a support matrix, (e) hybridizing recovered and blotted RNA with a labelled riboprobe specific for at least one of said genes, and (f) quantitatively determining the extent of binding of the labelled riboprobe to the blotted RNA. The use of varying induction processes on separate aliquots of a cell sample allows an understanding to be obtained of the dynamic functionality of the said gene or genes in the cells.