Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes.

Abstract

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides. Increasing formaldehyde concentration in the fixative decreases the signal intensity; 1% formaldehyde fixation provides the most intense signal. Several controls, including those with addition of unlabeled oligonucleotides to the hybridization buffer, confirm the specificity of mRNA detection. The sensitivity of the biotinylated probes is identical or lower as compared to the corresponding radiolabeled oligonucleotides. Histological and subcellular resolution is greatly enhanced with biotinylated probes. The rat vasopressin probes stain magnocellular neurons in the supraoptic and paraventricular nuclei and, under optimal conditions, parvocellular neurons in the suprachiasmatic nucleus. Vasopressin mRNA is present in the cytoplasm of the cell bodies and in the roots of certain processes. Calcitonin and calcitonin gene-related peptide mRNA are found co-localized in the cytoplasm of the same tumor cells in human medullary thyroid carcinoma.

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

“…In our hands, however, protease digestion resulted in an increase in signal intensity (Fig. 4), indicating that the unmasking of target mRNA by proteolysis is still important even in using oligo-DNA probe, in agreement with the previous reports (15,25). The discrepancy may be due to the difference in the fixatives used.…”

“…Recently, two groups (15,25) have reported the use of biotinylated oligo-DNA probes for in situ hybridization. Both groups obtained good results using the oligo-DNA labelled in the 3'-end with biotin-1 1 -dUTP by use of terminal deoxynucleotidyl transferase and using avidin-alkaline phosphatase system for signal detection.…”

“…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).…”

“…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). Recently we have developed a new method using thymine-thymine (T-T) dimerized DNA as a non-radioactive probe (21,22,33), which is thought to be the simplest in terms of no requirement for a separation of labelled DNA from unreacted labelling.…”

Immunohistochemical detection of human cellular yes gene (c-yes-1) messenger RNA by a non-radioactive synthesized oligodeoxynucleotide probe.

“…The most efficient probes are cDNAs or cRNAs into which biotinylated nucleotides are incorporated and are secondarily revealed by using a histochemical or an immunohistochemical reaction. Development of non-radioactive probes for ISH is profitable because they have a long shelf life, they avoid the biohazards and the autoradiographic procedure linked to the use of radio-isotopes, they give optimal histological resolution, and they provide results 549 within hours after the completion of ISH (7,18).…”