A branched DNA signal amplification assay to quantitate messenger RNA of human uncoupling proteins 1, 2, and 3.
Uncoupling proteins (UCP) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. Three subtypes of UCP have been identified so far. The regulation of UCP expression is mainly controlled at the transcriptional level, thus making the measurement of UCP mRNA beneficial for both diagnosis and research of weight disorders and diabetes. We have developed an assay using the branched DNA signal amplification assay (bDNA assay) to quantitatively measure the mRNA levels for human UCP1, 2, and 3. UCP-subtype-specific primers were designed for the assay. RNA transcripts of each UCP generated by in vitro transcription were used to validate the specificity and sensitivity of the assay. The quantitative measurement of UCP mRNA was further demonstrated with cultured cells and human tissue. A comprehensive survey of UCP expression from 17 human tissues measured by the newly developed assay is provided. The method described here offers a rapid, sensitive, specific, and quantitative assay for measurement of human UCP mRNA.
Zhou
L
L
Endocrine Therapeutics, Drug Discovery, The R.W. Johnson Pharmaceutical Research Institute, 1000 Route 202, Raritan, New Jersey 08869, USA. lzhou@prius.jnj.com
Cryan
E V
EV
Minor
L K
LK
Gunnet
J W
JW
Demarest
K T
KT
D017667:Adipocytes / Q000737:chemistry; D000273:Adipose Tissue / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D005821:Genetic Techniques; D006801:Humans; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000737:chemistry; D024101:Mitochondrial Proteins; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D014158:Transcription, Genetic; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3
D002352:Carrier Proteins; D017931:DNA Primers; D007473:Ion Channels; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D012333:RNA, Messenger; C000604371:UCP1 protein, human; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D000071247:Uncoupling Protein 3; D004247:DNA