A one-step procedure for isolation of poly(A)+ mRNA from isolated brain capillaries and endothelial cells in culture.
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.
Boado
R J
RJ
Department of Medicine and Brain Research Institute, UCLA School of Medicine 90024-1682.
Pardridge
M M
MM
D000818:Animals; D015152:Blotting, Northern; D002196:Capillaries / Q000737:chemistry; D002417:Cattle; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology; D015870:Gene Expression; D005947:Glucose / Q000172:deficiency; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D011061:Poly A / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*
D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D011061:Poly A; D005947:Glucose