Method for simple analysis of relative nucleic acid levels in multiple small samples by cytoplasmic dot hybridization
US4677054A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Aug 8, 1983 |
| Grant date | Jun 30, 1987 |
| Priority date | — |
| Expiry date | Aug 8, 2003 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Q1/6841
- WIPO fieldBiotechnology
- WIPO sectorChemistry
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 wh…
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