Measurement of Blood‐Brain Barrier GLUT1 Glucose Transporter and Actin mRNA by a Quantitative Polymerase Chain Reaction Assay

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Abstract
The expression of the blood-brain barrier GLUT1 glucose transporter is down-regulated in brain capillary endothelial cells in tissue culture. Consequently, the study of the regulation of this low-abundance transcript requires the isolation of poly(A)+ mRNA from relatively large numbers of brain endothelial cells in culture (approximately 10(7)). Therefore, in order to facilitate studies with smaller amounts of cells, we describe here a quantitative polymerase chain reaction (PCR) assay to measure the mRNA of GLUT1 and the mRNA of the housekeeping gene, actin, which is used as standard control. Bovine brain endothelial cells were grown as either a primary culture (EP cells) or as a brain endothelial cell line (ECL cells) in 25-mm 6-well cluster dishes, and total or poly(A)+ RNA was isolated. Following synthesis of cDNA with AMV reverse transcriptase and oligo(dT)18 primer, PCR was performed with sense and antisense primers for bovine GLUT1 and gamma-actin, respectively. Reactions were performed in the presence of 2.5 microCi of [alpha-32P]dCTP, and products were resolved in agarose gels and quantified by scanning densitometry of autoradiograms. A direct relationship between RNA-cDNA and PCR products was observed for GLUT1 after 30 cycles, and for actin after 15 PCR cycles. The method was reproducible within specified ranges of starting RNA-derived cDNA, and the intraassay coefficient of variation averaged 7.2 +/- 1.8%. The GLUT1/actin mRNA ratio was as follows: brain capillaries >> EP > ECL. In addition, it is demonstrated that tumor necrosis factor-alpha induced a three- to fourfold increase in the GLUT1/actin mRNA ratio in ECL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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