In situ hybridization; principles and applications: review article

In situ hybridization (ISH) is a method that uses labeled complementary single strand DNA or RNA to localize specific DNA or RNA sequences in an intact cell or in a fixed tissue section. The main steps of ISH consist of: probe selection, tissue or sample preparation, pre-hybridization treatment, hybridization and washing, detection and control procedure. Probe selection is one of the important aspects of successful hybridization. ISH sensitivity and specificity can be influenced by: probe construct, efficiency of labeling, percentage of GC, probe length and signal detection systems. Different methods such as nick translation, random priming, end tailing and T4 DNA polymerase replacement are used for probe generation. Both radioactive and non-radioactive labels can be used in order to probe labeling. Nucleic acid maintenance in samples, prevention of morphological changes of samples and probe penetration into tissue section are the main aims of sample preparation step. Then, a small amount of solution containing probe, is added on slides containing tissue sections for hybridization process, then slides are incubated overnight. Next day, washes are carried out to remove the probes which are not bound to target DNA or RNA. Finally, in order to be sure that the observed labeling is specific to the target sequence, using several control procedures is very important. Various techniques based on ISH consist of: Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), genomic in situ hybridization (GISH), comparative genomic hybridization (CGH), spectral karyotyping (SKY) and multiplex fluorescence in situ hybridization (MFISH). One of the most common techniques of ISH is fluorescence in situ hybridization. FISH can be used to: 1) detect small deletions and duplications that are not visible using microscope analysis, 2) detect how many chromosomes of a certain type are present in each cell and 3) confirm rearrangements that are suspected after microscope analysis. In this technique different fluorescent labels are attached to the probes. In this review article ISH, its different types, their application, advantages and disadvantages have been considered.