IN-SITU HYBRIDIZATION TO RNA

The pattern of RNA distribution in tissues and embryos can be studied directly by in situ hybridization. This chapter describes basic protocols that deal with the principles of RNA hybridization and discusses various probe preparation methods and specific applications for detection of multiple products. The sensitivity and quality of RNA in situ hybridization critically depends on the condition used for fixation and pretreatment of embryos. Before fixation, the chorion and the vitelline membrane are removed from embryos. Dechorionation is achieved by treating embryos with commercial bleach. Devitellinization is achieved by osmotic shock by transferring embryos from a heptane/formaldehyde interface to methanol. The efficiency of devitellinization depends on the age and sometimes on the maternal genotype. Hybridization conditions are chosen to preserve tissue morphology and allow sensitive annealing of the probe to the target RNA. Formamide is included in the hybridization solution to allow for lower hybridization temperatures while maintaining high stringency of hybridization. The methods described in the chapter are used for visualizing gene products in Drosophila embryos but applications for imaginal discs have also been described. © 1994, Elsevier Science Publishers, B.V. All rights reserved.