In Situ Hybridization: The Importance of Ultrapure Water for RNA Technologies

Abstract

Principle of In Situ Hybridization ISH represents an alternative to immunohistochemical staining if adequate antibodies are not available, and is employed in diverse areas of research. This type of hybridization uses specific nucleic acid fragments (probes) that are complementary to the target sequence in order to detect specific transcripts. Such probes may consist of DNA or of RNA. Meanwhile, RNA probes are frequently utilized. Such probes are commonly labeled with the molecule digoxigenin (DIG), which normally occurs in the plant species Digitalis purpurea. DIG labeling enables the specifically bound probes in a tissue section to be visualized by enzyme-conjugated anti-DIG antibodies. For this purpose, after incubation of tissue sections with antibody, the appropriate substrate is pipetted onto the sections and converted by an immunoenzymatic reaction into a visible colorant (Figure 1). This method can be used to analyze the activity of specific genes for research projects or diagnostic procedures. Further practical information on ISH is provided in Wilcox‘s overview article.[2] This article discusses the results of the ISH that was carried out as part of a cancer research project. The analyzed skin samples were obtained from genetically modified mice. Based on targeted manipulation of the tumor suppressor gene Patched (Ptch) by homozygous knockout, the Ptch-knockout mice developed basal cell carcinomas.[3] These skin tumors are the ones that most commonly occur in humans. They frequently exhibit increased activity of the signaling pathway that is regulated by Ptch. Inactivation of this important component in this mouse model pathologically activates the signaling pathway. This results in an increased expression of the target gene Gli1 — a transcription factor that activates a variety of other genes — in tumor cells, and expression of Gli1 can be detected using ISH.