METHOD FOR SIMPLE ANALYSIS OF RELATIVE NUCLEIC ACID LEVELS IN MULTIPLE SMALL SAMPLES BY CYTOPLASMIC DOT HYBRIDIZATION

摘要

? 280 HRL/JEL 8/2/83 A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numberous small samples of biological specimens is described. The technique involves denaturation of cytoplasmic preparations, followed by dotting of up to 96 samples onto a single sheet of nitrocellulose, hybridization with a 32P-labeled cDNA plasmid, autoradiography, and scanning. By analyzing cytoplasmic preparations instead of purified RNA, manipulations of multiple samples prior to analysis are minimized. Experiments with a clonal line of rat pituitary tumor (GH3) cells show that this technique can be employed to follow the induction by Ca2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 2.5 x 104 cells. The specificity of the technique for prolactin mRNA is shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences. Experiments with cultured rat hemipituitaries show that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary can be readily detected. This technique is quite simple, can be quantified, and permits the ? 280 HRL/JEL 8/2/83 simultaneous analysis of multiple samples while requiring very small amounts of material for analysis. Hence, it should be quite useful for example for studies with various experimental systems of the regulation of specific mRNA levels.