In situ hybridization (ISH)In situ hybridization (ISH) is a unique molecular analysis method providing accurate localization of endogenous, bacterial or viral nucleic acids such as DNA, mRNA, and microRNA in metaphase spreads, cells, and tissue preparations. Fixation and pre-treatment of samples are followed by hybridization of a labeled probe by complementary base pairing to the target and detection of the label to allow microscopic visualization of the hybrid. Detection of the probe can be achieved by chromogenic or fluorogenic techniques referred to as chromogenic in situ hybridization (CISH) or fluorescent in situ hybridization (FISH), respectively. One common application is the detection of genetic aberrations in cancer and in prenatal/postnatal samples. ISH probe generation techniques Because hybridization can take place between complementary deoxyribonucleotides or ribonucleotides, either DNA- or RNA-based probes (riboprobes) can be used to localize DNA or RNA in a given sample. Commercially available probes are expensive and do not always offer experimental flexibility as they come with a single label type. Therefore, many
researchers still heavily rely on preparing their own FISH probes by using one of the following techniques: nick translation or random-primed labeling for generation of long, double-stranded DNA probes, terminal-labeling of oligonucleotides and in vitro transcription from vectors containing RNA polymerase promotors to produce riboprobes. ISH probe detection techniques Direct detection is possible thanks to fluorescent labels that can be introduced during FISH probe
synthesis and detected by fluorescence microscopy. Multiplexing can easily be envisaged as two or more different probes labeled with different fluorophores can be visualized at any single time. Enzo offers fluorophores with several distinct colors to choose from, spanning the visible light spectrum and guaranteeing high signal intensity and good photostability. This direct approach provides a simple and efficient method to label multiple sequences for FISH. Multiplex DNA FISH can be used to
identify genetic rearrangements (e.g. ALK, BCR-ABL, HER2, MYB) while multiplex RNA FISH allows the generation of expression profiles, both in time and in space, of several mRNA targets. Enzo offers a complete set of solutions for labeling, hybridization, and detection. Check out our Genomics platform for more information and our Successful Research Tips. Additionally, contact our Technical Support Team for further assistance. |