Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

Principle FISH was developed in the early 1980s. 56 It applies fluorescent-labeled short DNA probes that hybridize to target DNA or RNA sequences in situ and fluorescence microscopy to localize and detect the targets in tissue slides. FISH can determine the presence or absence of mRNA expression from a gene of interest, as well as localize these gene expressions in specific cells. 57, 58 Pre-treatment is required to preserve RNA and tissue morphology. Cells, FFPE, or frozen tissue sections are fixed, then permeabilized to allow target accessibility. A target-specific probe hybridizes to the target RNA(s). Separate but compatible signal amplification systems enable the multiplex assay. Signal amplification is achieved via series of sequential hybridization steps. Multicolor FISH can be used to identify as many labeled features as there are different fluorophores used in the hybridization. 57, 58 Probe design Many different types of probes are used for in FISH, including cDNA, cRNA, and synthetic oligonucleotide probes. Probe size is important because longer probes hybridize more specifically than shorter probes, so that strands of DNA or RNA (often 20-50 nucleotides), which are complementary to a given target sequence, are often used to locate a target. 57, 58, 59 Data analysis Automated fluorescence signal analysis system can analyze fluorescence signal patterns / FISH spots in cells or cell nuclei automatically, precisely, and reproducibly. Signal channels can be combined to an assay, and the images obtained in each channel can be processed individually. Morphology of target cells, nuclei, or other objects can be precisely defined with a number of cell selection parameters. Multiple features of objects (e.g. area, shape, intensity, and signal distribution) can be measured. 57