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A One‐Step Procedure for Isolation of Poly(A) + mRNA from Isolated Brain Capillaries and Endothelial Cells in Culture
Author(s) -
Boado Ruben J.,
Pardridge William M.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb06433.x
Subject(s) - northern blot , messenger rna , choroid plexus , microbiology and biotechnology , biology , cell culture , endothelial stem cell , blood–brain barrier , chemistry , biochemistry , in vitro , gene , endocrinology , central nervous system , genetics
The study of the regulation of low‐abundance blood‐brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A) + mRNA. Therefore, we describe here a single‐step method for isolation of polyfA) + mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo‐de‐oxythymidine cellulose affinity chromatography. The yield of poly(A) + mRNA was—15‐19 #g/g of brain or choroid plexus, 14‐17 μg per batch of isolated capillaries in a single bovine forebrain (190 g), and 6‐12 μg/10 7 ECL cells. Northern blot analysis showed characteristic and undegraded 2.1‐and 1.7‐kb actin transcripts in brain capillaries and a 2.1‐kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up‐regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two‐step method.