Quantitative reverse transcriptase-polymerase chain reaction of glucose transporter 1 mRNA levels in rat brain microvessels.
We investigated the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify glucose transporter 1 (GLUT1) mRNA in cerebral microvessels. The technique was validated using an in vitro-transcribed RNA fragment (riboprobe) of partial 3' noncoding sequence of rat brain GLUT1 gene. A known amount of the riboprobe was reverse-transcribed to cDNA (target DNA). PCR primers were made to amplify a 292-bp fragment of the target DNA. The 5' primer was end labeled with 32P. An oligonucleotide of 100 bp containing the same sequences as the first 30 and the last 70 bases of the 292-bp fragment of the target DNA was synthesized and used as competitive DNA. The target DNA was coamplified with increasing amounts of competitive DNA using the same two primers. The ratio of radioactivity between amplified products of the target DNA (292-bp fragment) and the competitive DNA (100-bp fragment) was determined quantitatively after separation by gel electrophoresis and radioactivity counting. This method gave an accurate estimation of the amount of the riboprobe in the reaction and a 2- to 5-fold change in the amounts could be detected. By this method, the mean amount of GLUT1 mRNA from purified rat brain microvessels was estimated to be 1.5 +/- 0.1 x 10(-6) ng/ng total RNA. This value was about 10-fold higher than that in rat cell line PC12.
Wadhwani
K C
KC
Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892.
Fukuyama
R
R
Giordano
T
T
Rapoport
S I
SI
Chandrasekaran
K
K
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000098:blood supply* / Q000502:physiology*; D004247:DNA / Q000032:analysis / Q000235:genetics; D005784:Gene Amplification; D008833:Microcirculation / Q000737:chemistry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D016716:PC12 Cells; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results
D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase