Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle.
Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system.
Lengacher
Sylvain
S
Institut de Physiologie, Université de Lausanne, Switzerland.
Magistretti
Pierre J
PJ
Pellerin
Luc
L
D002001:Adipose Tissue, Brown / Q000737:chemistry*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D002540:Cerebral Cortex / Q000737:chemistry*; D015870:Gene Expression; D007473:Ion Channels; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D051379:Mice; D024101:Mitochondrial Proteins / Q000235:genetics; D000071245:Mitochondrial Uncoupling Proteins; D018482:Muscle, Skeletal / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014475:Uncoupling Agents / Q000032:analysis*; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D000071245:Mitochondrial Uncoupling Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; C117577:SLC25A27 protein, human; C492124:Slc25a14 protein, mouse; C000604371:UCP1 protein, human; C000604392:UCP3 protein, human; C000604372:Ucp1 protein, mouse; C000604393:Ucp3 protein, mouse; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3