In Situ Hybridization tor RNA: Nonradioactive Probe: Oligo-DNA Probe: Digoxigenin (II)
Nonradioactive in situ hybridization is thought to be less sensitive than the radioactive method, especially when used with oligo- DNA probe. However, the sensitivityofnonradioactive in situ hybridization using oligo-DNA is sufficient for localization of mRNA at cellular level even in human biopsy specimens. The method of nonradioactive in situ hybridization was developed in our laboratory in 1994 (Miyazaki et al. 1994) and was applied to identify and localize mRNA of several factors synthesized by renal cells in normal individuals and patients with a variety of renal diseases (Suzuki et al. 1995; Miyazaki et al. 1996). Nonradioactive in situ hybridization with oligo-DNA has several advantages compared with the radioactive method, including a high resolution, good probe stability and no exposure to radiation, as described in detail by Koji and Nakane (1996). Compared with other types of probes, such as RNA or cDNA probe, oligoDNA has several benefits. First, oligo-DNA is easier to prepare. While several years ago, a special DNA synthesizer that was available only in certain laboratories, was necessary for the preparation of oligo-DNA, such synthesizers are less expensive nowadays. Alternatively, custom made probes can be purchased nowadays from several commercial sources.
KeywordsStandard Saline Citrate Endogenous Biotin Normal Swine Serum Prehybridization Buffer Radioactive Method
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