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A Total Extract Dot Blot Hybridization Procedure for mRNA Quantitation in Small Samples of Tissues or Cultured Cells

Tipo de material: TextoTextoSeries ; Analytical BioChemistry, 172(2), p.436-443, 1988Trabajos contenidos:
  • Grimes, A
  • Mcardle, H.J
  • Mercer, J.F.B
Tema(s): Recursos en línea: Resumen: A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg)or cultured cells (lower limit IO5 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 ofthe 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.
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A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg)or cultured cells (lower limit IO5 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 ofthe 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.

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