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 poly(A)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo-deoxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was approximately 15-19 micrograms/g of brain or choroid plexus, 14-17 micrograms per batch of isolated capillaries in a single bovine forebrain (190 g), and 6-12 micrograms/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.
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