Rapid chemiluminescent detection of mRNAs on northern blots with digoxigenin end-labelled oligonucleotides.
A simplified, nonradioactive procedure for the detection of specific mRNAs on Northern blots has been developed, utilizing digoxigenin-labelled oligonucleotides and chemiluminescence. Antisense oligonucleotide (30-35 mer) probes were designed and synthesised based on published cDNA and gene sequences. These probes were end-labelled (5') with digoxigenin. Total RNA was fractionated by agarose gel electrophoresis and capillary blotted onto positively charged nylon membranes. After hybridization, the mRNA/digoxigenin-labelled oligonucleotide complex was detected by a chemiluminescence-based method using disodium 3-(4-methoxyspiro-[1,2-dioxetane-3-2'(5'chloro)- tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate (CSPD) as substrate. The advantages of this simplified technique for detecting mRNAs in physiological and nutritional studies are described.
Trayhurn
P
P
Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, UK.
Duncan
J S
JS
Nestor
A
A
Thomas
M E
ME
Eastmond
N C
NC
Rayner
D V
DV
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods*; D004076:Digoxigenin; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D008071:Lipoprotein Lipase / Q000235:genetics; D008163:Luminescent Measurements; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D009124:Muscle Proteins; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D008071:Lipoprotein Lipase; D004076:Digoxigenin