Chromogenic in situ hybridization
[1][2] It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification.
[1] CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA.
Only a few CISH probes are available commercially, so for most applications they have to be extracted, amplified, sequenced, labelled and mapped from bacterial artificial chromosomes (BACs).
DNA is extracted from the BAC clones and amplified using a polymerase-based technique, such as degenerate oligonucleotide primed (DOP)-PCR.
[9] Probe labelling can be carried out by using either random priming or nick translation to incorporate biotin or digoxigenin.
[11] As a final step, 10–20 μL of probe is added, the sample is covered with a coverslip which is sealed with rubber cement, and the slide is heated to 97 °C for 5–10 minutes to denature the DNA.
[1] If biotin was used as a probe label, non-specific binding sites must first be blocked using bovine serum albumin (BSA).
[6][11] HRP then converts diaminobenzidine (DAB) into an insoluble brown product, which can be detected in a bright-field microscope under 40- to 60-fold magnification.
[5] FISH is considered to be the gold standard for the detection of chromosomal abnormalities because it is very sensitive and has high resolution.
[3][14] Other techniques that are developed to detect chromosomal abnormalities are usually compared to the sensitivity and specificity of FISH to see how they measure up.
[3] Most other sources agree and report an almost equal performance on gene amplification assays for FISH and CISH.
[14] With regard to the overall method, FISH can be performed using direct labelling—fluorochromes are attached to the probes—or indirect labelling—the probes are labelled with biotin or digoxigenin which are then detected using fluorescently-labelled streptavidin or antibodies, respectively.
[14] CISH is performed using indirect labelling in which antibodies or streptavidin are conjugated to enzymes such as HRP or alkaline phosphatase (AP).
[18] CISH is frequently applied to assess gene amplification, such as HER-2/neu status in breast cancer samples.
ALK-positive tumors are a clinically relevant subgroup as they can be very effectively treated with the ALK inhibitor crizotinib.
