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

1994; Wiley; Volume: 62; Issue: 6 Linguagem: Inglês

10.1046/j.1471-4159.1994.62062085.x

1471-4159

Rubén J. Boado, William M. Pardridge,

RNA modifications and cancer

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 (˜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 7‐actin, respectively. Reactions were performed in the presence of 2.5 μCi of [α‐ 32 P]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 β 1.8%. The GLUT1/actin mRNA ratio was as follows: brain capillaries β EP > ECL. In addition, it is demonstrated that tumor necrosis factor‐α induced a three‐to fourfold increase in the GLUT1/actin mRNA ratio in ECL cells. This method provides a 100‐200‐fold increase in the sensitivity of detection of blood‐brain barrier GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture compared with the conventional northern blotting technique using poly(A) + mRNA.