Use of nucleotides as an alternative to formamide in non-radioactive in situ hybridization.

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To analyze the expression of specific mRNA at the level of individual cells, non-radioactive in situ hybridization has been a most powerful technique. In the process of in situ hybridization, the use of formamide is usually required in order to reduce the melting temperature (Tm) of nucleic acids. However, formamide is an expensive and unstable reagent, and more importantly, formamide in itself has some deteriorative effects such as nonspecific staining and morphological damage on the results of in situ hybridization. In this study, we examined the use of a mix-ture (Nm) of nucleotides (AMP, GMP, UMP, CMP) as an alternative to formamide in our non-radioactive hybridization system with thymine-thymine (T-T) dimerized DNA probe. When the effects of Nm on re-annealing of denatured pBR 322 DNA were investigated by electrophoretic patterns on agarose gel, it was confirmed that Nm (about 20-200mg/ml) reduced the Tm of pBR 322 DNA. On dotblot hybridization using Nm, we obtained sensitive and specific results similar to that of formamide. Finally, on frozen sections of rat pituitary glands fixed by perfusion of 4% paraformaldehyde in phosphate buffered saline (pH 7.4), prolactin mRNA was successfully localized in situ using Nm instead of formamide.

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