A One‐Step Procedure for Isolation of Poly(A)+ mRNA from Isolated Brain Capillaries and Endothelial Cells in Culture

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Abstract
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/107 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.

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