Measurement of GLUT mRNA in liver of fetal and neonatal rats using a novel method of quantitative polymerase chain reaction.
Transfer of glucose into the hepatocyte is mediated by glucose transporters (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 semiquantitative and has no internal control during first-strand synthesis. We designed a method of coreverse transcription and PCR amplification using bovine rhodopsin as an internal control for both cDNA synthesis and amplification. As part of the validation of this technique, we determined 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 were no secondary structure effects on amplification. We applied our modified method of RT-PCR to measure the ontogeny of GLUT expression in liver of fetal and postnatal rats (d20 fetuses and d1, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 increased 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 provides an internal control and allows measurement of mRNA levels in small quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low.
Lane
R H
RH
Division of Neonatology, Northwestern University Medical School and Children's Memorial Hospital, Chicago, Illinois, 60614, USA.
Flozak
A S
AS
Simmons
R A
RA
D000818:Animals; D000831:Animals, Newborn; D002417:Cattle; D005260:Female; D005333:Fetus / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012243:Rhodopsin / Q000378:metabolism; D014158:Transcription, Genetic
D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D012243:Rhodopsin