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 detergents are used to break down the lipids.

The target RNA is then hybridized with the probes at a temperature just below the melting point of the RNA-probe hybrid. This helps the probes to anneal with the complementary mRNAs. Unbound and loosely bound probes are removed by performing several washes.

The method for detecting the hybridized probes depends on the type of label. Radioactive probes are exposed to photographic films, while fluorescent probes are visible under fluorescent microscopes.

For other labels, antibodies against the reporter molecule are tagged with a fluorescent or colorimetric dye.

Detection of hybridized probes reveals the distribution of specific mRNAs indicating where the genes of interest are expressed.