Histochemical detection of the messenger RNAs coding for calcitonin and calcitonin gene-related peptide in medullary thyroid carcinomas with radioactive and biotinylated oligonucleotide probes.
The present study has been undertaken to investigate the efficiency of biotinylated synthetic oligonucleotide probes in detection by in situ hybridization of the mRNAs coding for calcitonin (CT) or calcitonin gene-related peptide (CGRP) in human medullary thyroid carcinomas (MTCs). Tissue sections fixed with formaldehyde were hybridized with 45-base long oligonucleotides, specific for CT or CGRP mRNA. Recombinant DNA probe or synthetic oligonucleotides radioactively labelled with 32P or 35S were used as controls to detect by autoradiography the corresponding mRNAs in the tumour cells. Oligonucleotide probes labelled by fixation of one biotin molecule at their 5'-end, or by incorporation of a tail of biotin-11-dUTP at their 3'-end, were used and were revealed by incubation with streptavidin-alkaline phosphatase associated with the corresponding substrate. Each biotinylated probe stained exclusively the cytoplasm of the tumour cells, the CT probe giving a much higher level of staining than the CGRP probe. The same cells were found to contain CT and CGRP mRNAs. Controls performed with either radioactive or biotinylated probes confirmed the specificity of the staining. These results demonstrate that biotinylated synthetic oligonucleotides can be used as efficient tools to investigate gene expression in tissue sections, thus avoiding the various inconveniences connected with the use of radioactive probes, especially bio-hazards, the use of autoradiography, the limited histological resolution, and the delay in obtaining results.
Mougin
C
C
Laboratoire de Physiologie, Faculté de Médecine, Besançon, France.
Guitteny
A F
AF
Fouque
B
B
Viennet
G
G
Teoule
R
R
Bloch
B
B
D001345:Autoradiography; D001710:Biotin; D002116:Calcitonin / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000032:analysis*; D013462:Sulfur Radioisotopes; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology
D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D001710:Biotin; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide