Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Abstract

Synthetic oligonucleotide probes can be easily obtained and used, in contrast to cDNA cloning to develop probes, and thus the present study was carried out to determine whether such probes could also be useful for in situ hybridization. A 24-base synthetic oligonucleotide complementary to part of the a-melanocyte-stimulating hormone (a-MSH) coding region of proopiomelanocortin (POMC) mRNA was 5'-end-labeled by using [y-32P]ATP with T4 polynucleotide kinase or was 3' tailed by using [a-32P] (200-250 g; Charles River Breeding Laboratories) were anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused intracardially with 50 ml of ice-cold saline followed by cold 0.1 M phosphate-buffered 4% formaldehyde for 30 min (21). One group of animals (n = 6) had been treated with haloperidol (2 mg/kg per day, i.p.) or saline for 4 days prior to perfusion. Pituitaries were removed, postfixed in ice-cold buffered formaldehyde for 1 hr, and incubated overnight in 15% sucrose in 0.02 M phosphate-buffered saline (21) at 4°C. The tissues were then frozen in liquid N2, and 10-,um-thick sections were cut in a cryostat at -20°C, thaw-mounted onto gelatin-coated slides, and stored at -80°C.Probes

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

“…Primer Extension Assay-Primer 5Ј-ACCCCGGATCCGC-GCCCACGGCAGATAGG-3Ј was labeled with [␥-32 P]dATP at 37°C using T4 polynucleotide kinase (Fermentas) for 1 h by the 5Ј-end labeling method (22). The labeled primer was incubated with the cross-linked RNA-protein or RNA-6AP/6APi complex (ϳ10 g) at 65°C for 5 min.…”

Detection of RET oncogene activation in human papillary thyroid carcinomas by in situ hybridisation

“…The probes were purified by polyacrylamide gel electrophoresis and dissolved in 10 mM Tris, 0.1 mM EDTA at a concentration of 1 fgtI-'. They were labelled at the 3 min terminus with 35S (9.25 MBq, 10mCiml-1, Amersham) using terminal dioxynucleotidyl transferase (TdT, 50 U; Boerhinger Mannheim) according to Lewis et al (1986). The labelled probes were purified using a G25 Sephadex quick spin column (Boerhinger Mannheim).…”

In situ localization of c-myc mRNA in HL-60 cells using non-radioactive synthetic oligodeoxynucleotide probes.

“…Among papers published previously (7,12,15,25,28,34,36,44,45), various conditions concerning hybridization and washing were employed in in situ hybridization with synthetic oligo-DNA probes. These situations were made more complicated by the fact that the theoretical formula does not always work in this area (2).…”

“…Most of the studies with oligo-DNA probes used 32P-labelling (12,28,34,36,44) or 1251-labelling (7) for signal detection though the resolution was not always sufficient for the analysis of individual cells. Recently, in order to obtain a better resolution and to minimize the cumbersome procedures associated with the handling of radioactive compounds, non-radioactive labels have been developed (13,19,26,35,42), and one of them has been applied to the in situ hybridization with oligo-DNA probe (15,25).…”