In situ hybridisatie

In situ hybridization is a technique used to locate DNA or RNA in cultured cells or in chemically preserved tissue sections.

RNA in situ hybridization is used to monitor where a gene is being expressed by detecting and quantifying the specific mRNA transcripts.

The target mRNAs are detected by single-stranded, complementary RNA probes that are created by in vitro transcription of a DNA template or synthesized by a chemical reaction.

The probes are labeled with radioactive isotopes, fluorophores, or other reporter molecules, such as biotin or digoxigenin, that can be bound and detected by a labeled antibody.

RNA in situ hybridization has four main steps: tissue fixation, permeabilization, hybridization, and detection.

To begin, fresh tissue is harvested and chemically treated to stop any biochemical reactions within the tissue and preserve its structural integrity. This preservation technique is called tissue fixation.

Next, proteins and lipids in the tissue need to be removed for the probe to access and bind the target RNA.

To degrade the proteins and permeabilize the cell membrane, the sample is treated with 0.2 molar hydrochloric acid and enzymes such as proteinases, while deterg