A non-radioactive method to detect RNA or DNA using an oligonucleotide probe with bromodeoxyuridine free ends, a monoclonal antibody against bromodeoxyuridine and immunogold silver staining.
A non radioactive method for probing RNA or DNA on dot and Northern blots using a synthetic oligonucleotide with bromodeoxyuridine free ends is described. The present experiment was carried out with human testis and placental RNA's. The probe was the 21 base long sequence coding for the amino acids 18 to 24 of the insulin-like growth factor I (IGF-I) with two bromodeoxyuridine dinucleotides added at the 5' and 3' ends. The probe was detected with a monoclonal antibody against bromodeoxyuridine and immunogold silver staining (IGSS). Our method was compared to the peroxydase (HRP) revelation of the same probe. The results obtained show a lower background with IGSS than with HRP revelation. A sensitivity similar to that of 32P labelling was found with the advantages of an increase in the rapidity of the procedure (24 hours instead of 9 days exposure) and the absence of handling radioactive substances. Moreover, as the monoclonal antibody against BrdU detects single stranded DNA only, the use of BrdU free ends-labelled oligonucleotide allows the development of the revelation procedure without any previous denaturation of the hybrid. This particular point is an indisputable advantage for detecting hybridization in situ.
Scippo
M L
ML
Institut de Pathologie, Centre Hospitalier Universitaire, Liège, Belgique.
Dombrowicz
D
D
Igout
A
A
Closset
J
J
Hennen
G
G
D000911:Antibodies, Monoclonal; D001345:Autoradiography; D015152:Blotting, Northern; D001973:Bromodeoxyuridine / Q000032:analysis*; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D007124:Immunoenzyme Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012313:RNA / Q000032:analysis*; D015347:RNA Probes
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D015345:Oligonucleotide Probes; D015347:RNA Probes; D012313:RNA; D004247:DNA; D001973:Bromodeoxyuridine