MEASUREMENT OF BLOOD-BRAIN-BARRIER GLUT1 GLUCOSE-TRANSPORTER AND ACTIN MESSENGER-RNA BY A QUANTITATIVE POLYMERASE CHAIN-REACTION ASSAY

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 tran script requires the isolation of poly(A)(+) mRNA from relatively large numbers of brain endothelial cells in culture (similar to 10(7)). The refore, 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, ac tin, which is used as standard control. Bovine brain endothelial cells were grown as either a primary culture (EP cells) or as a brain endot helial cell line (ECL cells) in 25-mm 6- well cluster dishes, and tota l or poly(A)(+) RNA was isolated. Following synthesis of cDNA with AMV reverse transcriptase and oligo(dT)(18) primer, PCR was performed wit h sense and antisense primers for bovine GLUT1 and gamma-actin, respec tively. Reactions were performed in the presence of 2.5 mu Ci of [alph a-P-32]dCTP, and products were resolved in agarose gels and quantified by scanning densitometry of autoradiograms. A direct relationship bet ween 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 coe fficient of variation averaged 7.2 +/- 1.8%. The GLUT1/actin mRNA rati o was as follows: brain capillaries >> EP > ECL. In addition, it is de monstrated that tumor necrosis factor-alpha induced a three- to fourfo ld increase in the GLUT1/actin mRNA ratio in ECL cells. This method pr ovides a 100-200-fold increase in the sensitivity of detection of bloo d-brain barrier GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture compared with the conventional northern blott ing technique using poly(A)(+) mRNA.