A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells
✍ Scribed by Andrew Grimes; Harry J. McArdle; Julian F.B. Mercer
- Publisher
- Elsevier Science
- Year
- 1988
- Tongue
- English
- Weight
- 596 KB
- Volume
- 172
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.