Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples

Abstract

A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numerous small samples of animal cells or tissue 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 is minimized. Experiments with a clonal line of rat pituitary tumor (GH3) cells showed 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 10(4) cells. The specificity of the technique for prolactin mRNA was shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences. Experiments with cultured rat hemipituitaries showed that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary could be readily detected. This technique is quite simple, requires very small amounts of cells or tissue, and permits the simultaneous analysis of multiple samples. Hence, it should be quite useful for studies with various experimental systems of the regulation of specific mRNA levels.