Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1983-08-08
1987-06-30
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
935 77, 935 78, 435 29, C12Q 168, C12Q 102, C12N 1500
Patent
active
046770542
ABSTRACT:
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 .sup.32 P-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 (GH.sub.3) cells show that this technique can be employed to follow the induction by Ca.sup.2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 2.5.times.10.sup.4 cells. The specificity of the technique for prolactin mRNA is shown by employing GC cells, a GH.sub.3 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 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.
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Bancroft F. Carter
White Bruce A.
Huleatt Jayme A.
Sloan-Kettering Institute for Cancer Research
White John P.
Wiseman Thomas G.
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