16.14: In-situ Hybridization
In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many different probes, such as single-stranded DNA probes, double-stranded DNA probes, antisense RNA probes or riboprobes, and synthetic oligodeoxynucleotide probes, are used in in situ hybridization. The choice of probe depends on several factors, including their sensitivity, specificity, stability, and ease of penetration into the tissue sample.
These probes can be labeled with radioisotopes, fluorescent dyes, or other antigen molecules for detection purposes. The 3H, 35S, and 32P are widely used radiolabeled probes, while non-radioactive labels include biotin, digoxigenin, and fluorescein. These labels can be attached to the probe DNA molecule via end-labeling, nick-translation, or random primer synthesis methods. Detection methods, such as autoradiography, fluorescence microscopy, or immunohistochemistry, are used for target visualization based on the label attached to the hybridized probe.
Advantages and disadvantages of in situ hybridization
One of the major advantages of in situ hybridization is that it can even be applied to frozen tissues to enable maximum use of tissues that are difficult to obtain. In addition, it can be combined with other techniques, such as immunohistochemistry, to detect protein and active mRNA in the sample. However, while working with samples that have low DNA and RNA copies, it may be difficult to identify targets using in situ hybridization.