MEASUREMENT OF GLUT MESSENGER-RNA IN LIVER OF FETAL AND NEONATAL RATSUSING A NOVEL METHOD OF QUANTITATIVE POLYMERASE CHAIN-REACTION

Transfer of glucose into the hepatocyte is mediated by glucose transpo rters (GLUTs). GLUT mRNA levels are usually measured by Northern blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method is only semi quantitative and has no internal control during first-strand synthesis . We designed a method of coreverse transcription and PCR amplificatio n using bovine rhodopsin as an internal control for both cDNA synthesi s and amplification. As part of the validation of this technique, we d etermined that there was no nonspecific amplification of bovine GLUTs by rhodopsin primers, that there were no differences in amplification due to different regions of the Glut gene amplified, and that there we re no secondary structure effects on amplification. We applied our mod ified method of RT PCR to measure the ontogeny of GLUT expression in l iver of fetal and postnatal rats (d20 fetuses and dl, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 inc reased with age. We were able to detect small quantities of GLUT 3 in fetal liver and of GLUT 5 in postnatal liver. This method of RT-PCR pr ovides an internal control and allows measurement of mRNA levels in sm all quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low. (C) 1996 Academic Press, Inc.