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A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells
A Grimes1, H J McArdle, J F Mercer
1Murdoch Institute for Research Into Birth Defects, Royal Childrens Hospital, Parkville Victoria, Australia.
Abstract:
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.