Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe.
A synthetic oligodeoxynucleotide sequence complementary to the mRNA for the adrenocorticotrophin (ACTH) precursor pro-opiomelanocortin (POMC) was end labelled using digoxigenin. The probe was used to detect POMC mRNA both on nitrocellulose filters and by non-isotopic in situ hybridisation (NISH) in tissue sections. Digoxigenin was identified using anti-digoxigenin alkaline phosphatase. The model system examined was the rat pituitary gland. Removal of both adrenal glands and dexamethasone administration were used to change the concentrations of POMC mRNA in the rat anterior lobe. The labelled probe reacted with a single band of appropriate molecular weight in Northern blot analysis. The distribution of signal in tissue sections and the changes induced by experimental manipulation were as predicted. The results indicate that this method of NISH will prove useful in the detection of specific messenger RNAs in tissue sections of buffered, formalin fixed, paraffin wax embedded material.
Farquharson
M
M
University Department of Pathology, Royal Infirmary, Glasgow.
Harvie
R
R
McNicol
A M
AM
D000818:Animals; D001710:Biotin / Q000032:analysis; D015152:Blotting, Northern; D004076:Digoxigenin; D004077:Digoxin / Q000031:analogs & derivatives; D008297:Male; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010902:Pituitary Gland / Q000032:analysis; D011333:Pro-Opiomelanocortin; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin; D001710:Biotin; D004077:Digoxin; D004076:Digoxigenin