Chromogenic In Situ Hybridization (CISH) is a molecular technique used to visualize specific DNA or RNA sequences directly within cells or tissue samples. It combines in situ hybridization with chromogenic detection, providing a visible, colored signal that marks the location of target genetic material.
Understanding the CISH Process
CISH uses a labeled probe complementary to the specific DNA or RNA sequence of interest. This probe binds to its target through hybridization. Tissue samples are prepared, fixed on slides, and their nucleic acids denatured to allow probe access.
After hybridization, an indirect detection method is used. CISH probes are labeled with molecules like biotin or digoxigenin, detected by antibodies or streptavidin conjugated to enzymes. Common enzymes include horseradish peroxidase (HRP) or alkaline phosphatase (AP).
Next, the enzyme converts a colorless substrate into a colored precipitate at the hybridization site. For example, HRP reacts with diaminobenzidine (DAB) to produce a brown precipitate. This colored product is visible under a standard brightfield microscope, allowing visualization of the target DNA or RNA sequence within the cell or tissue, preserving tissue morphology.
Where CISH is Used
CISH has broad applications in both diagnostics and research, offering a way to detect genetic alterations and infectious agents within tissue samples. One prominent application is in cancer diagnostics, particularly for detecting gene amplifications. For instance, CISH is widely used to assess the amplification status of the HER2 gene in breast cancer.
The HER2 gene, when amplified, indicates an aggressive form of breast cancer and helps guide treatment decisions, such as the use of trastuzumab therapy. CISH allows for the visualization of HER2 gene copies as distinct colored signals within the tumor cells, enabling pathologists to correlate gene status with the morphology of the tumor. This simultaneous evaluation of genetic information and tissue structure provides valuable insights for prognosis and treatment planning.
Beyond cancer, CISH is also employed in identifying viral infections within tissue samples. A notable example is the detection of Human Papillomavirus (HPV) in cervical biopsies and head and neck cancer. CISH can visualize the presence of HPV DNA or RNA, which is particularly useful for diagnosing HPV-related conditions and understanding the virus’s activity within the cells. The ability to visualize these targets directly within the tissue helps in understanding the spread and impact of the infection.
Comparing CISH to Other Techniques
CISH is often compared to Fluorescence In Situ Hybridization (FISH), another technique used for detecting nucleic acid sequences. A primary distinction lies in their detection methods: CISH uses chromogenic (color-producing) reactions, while FISH employs fluorescent probes. This difference dictates the type of microscopy required; CISH can be viewed using a standard brightfield microscope, which is commonly available in most pathology laboratories, whereas FISH necessitates a specialized fluorescence microscope.
One advantage of CISH is its ability to produce a permanent signal that does not fade over time, allowing for long-term archiving and re-examination of slides. This contrasts with FISH signals, which can diminish within weeks or months, requiring immediate digital recording. CISH also offers easier morphological correlation, as pathologists can simultaneously evaluate the genetic findings alongside the detailed tissue architecture under a brightfield microscope, a familiar viewing environment for them.
While CISH generally has a lower cost compared to FISH, it may have limitations in sensitivity for certain low-level gene expressions and less capability for multiplexing, meaning detecting multiple targets simultaneously with distinct colors, compared to FISH. FISH can more readily detect multiple targets using different fluorescent tags. However, CISH has dual-color assays available that can visualize two targets, such as HER2 and a chromosome 17 centromere reference, using different colored chromogens.