Chemiluminescent detection of mRNAs on northern blots with digoxigenin end-labeled oligonucleotides.
To establish a simplified, nonradioactive approach for identifying mRNAs on Northern blots, antisense oligonucleotides have been used as probes in combination with chemiluminescence-based detection. Oligonucleotides (approximately 32-mer) were end-labeled with digoxigenin (DIG) and used in conjunction with adamantyl 1,2-dioxetane aryl phosphate substrates (Lumigen PPD and CSPD). Oligonucleotides were designed as probes for several mRNAs in tissues of rats and mice, including the mitochondrial uncoupling protein, lipoprotein lipase, GLUT1, GLUT4, and beta-actin. Uncoupling protein mRNA was detected in total RNA from brown adipose tissue with a 32-mer DIG-labeled oligonucleotide, within 2 min of exposure to film. This mRNA could also be detected when as little as 250 ng of total RNA was applied to the gel, following 4 h exposure to film, and was present only in brown fat. The mRNA for lipoprotein lipase was detectable with a 30-mer DIG-labeled oligonucleotide in 1 micrograms of total RNA from mouse heart, within 2 h of exposure. The mRNA for the GLUT1 glucose transporter was detected in total RNA from rat midbrain using a 32-mer DIG-labeled oligonucleotide, while beta-actin mRNA was detected with a 30-mer oligonucleotide. The mRNA for the insulin-sensitive glucose transporter GLUT4 was detected with a 32-mer DIG-labeled oligonucleotide and found only in those tissues in which glucose uptake is stimulated by insulin. The speed of detection was greater with CSPD and was augmented by exposure of membranes to film at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, Scotland, United Kingdom.
P
P
Trayhurn
J S
JS
Duncan
A
A
Nestor
M E
ME
Thomas
D V
DV
Rayner
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics; D004076:Digoxigenin / Q000737:chemistry*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007473:Ion Channels; D008071:Lipoprotein Lipase / Q000235:genetics; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D009124:Muscle Proteins; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D000071256:Uncoupling Protein 1
D000199:Actins; D002352:Carrier Proteins; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C495678:Slc2a1 protein, mouse; C495679:Slc2a1 protein, rat; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D000071256:Uncoupling Protein 1; D008071:Lipoprotein Lipase; D004076:Digoxigenin