Rapid chemiluminescent detection of the mRNA for uncoupling protein in brown adipose tissue by northern hybridization with a 32-mer oligonucleotide end-labelled with digoxigenin.
Sequences for the uncoupling protein gene (or mRNA) expressed in brown adipose tissue have been re-examined for homology between species. A 32 bp region is identical in rats and cattle, this representing a 5 bp extension of the 27 bp conserved sequence previously described. In mice, rabbits, Syrian hamsters, and humans there is a single bp difference with the 32 bp conserved rat/cattle sequence. A 32-mer antisense oligonucleotide (3'-TAGTG-GAAGGGCGACCTGTGGCGGTTTCAGGC-5') targeted to the 32 bp rat/cattle sequence has been synthesized and end-labelled (3' and 5' ends) with digoxigenin, as a simple, non-radioactive probe for uncoupling protein mRNA. Hybridization of the oligonucleotide to the mRNA was readily detected on Northern blots with total RNA from brown adipose tissue of rats, mice, and newborn lambs, using a chemiluminescence substrate and following short exposure to film (1 h, or less). With longer exposure (overnight), uncoupling protein mRNA was detected in as little as 250 ng of total RNA from rat brown fat. The 32-mer digoxigenin-labelled oligonucleotide, coupled with a chemiluminescence substrate, provides a rapid, sensitive, non-radioactive procedure for detecting uncoupling protein mRNA on Northern blots, applicable across species.
Trayhurn
P
P
Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, Scotland, UK.
Duncan
J S
JS
D002001:Adipose Tissue, Brown / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics*; D006224:Cricetinae; D004076:Digoxigenin; D006801:Humans; D007473:Ion Channels; D008163:Luminescent Measurements; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008647:Mesocricetus; D051379:Mice; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D017385:Sequence Homology; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D000071256:Uncoupling Protein 1; D004076:Digoxigenin