mRNA DNA in situ hybridization
In situ hybridization (ISH) uses a labelled probe (complementary DNA, RNA or modified nucleic acids strand) to localize a specific DNA or RNA sequence in a portion or section of the tissue - in situ. DNA ISH can be used to determine the structure of chromosomes, and RNA ISH can be used to measure and localize mRNAs within individual cells in tissue sections.
The probes differ in terms of their labels and methods of detection. FISH probes are generally labelled with various fluorescent tags and must be detected under a fluorescence microscope, whereas CISH probes are labelled with biotin or digoxigenin and can be detected using a bright-field microscope. Analyses performed using conventional bright-field microscopes are easier to implement in diagnostic laboratories and is, therefore, our method of choice.
CISH was developed as an alternative to FISH for the more practical detection of the HER-2/neu oncogene amplification in breast cancer FFPE-material and is frequently applied to assess gene amplification. It has been shown that this gene amplification is of higher prognostic value than protein expression. CISH is also used for to detect chromosomal rearrangements and fusions, such as the fusion of the ALK tyrosine kinase domain in lung cancer. Apart from cancers, CISH has also been shown to be useful for detecting human papillomavirus infections.
FISH is the gold standard for detecting chromosomal abnormalities due to its high degree of sensitivity and high resolution. CISH has shown to be a comparable technique to FISH. Compared to FISH, CISH has been shown to have a sensitivity of 97.5% and a specificity of 94% for detection of HER-2/neu gene amplification. However, CISH sometimes shows lower sensitivity for low-level amplifications. In contrast to FISH, only a few CISH probes are available commercially, so for most applications they must be extracted, amplified, sequenced, labelled and mapped. Despite this limited availability of CISH probes, they are generally more cost-effective than FISH probes. It is worth noting that sensitive methods with signal amplification are constantly being developed. This is illustrated by the detection of Prostate cancer antigen 3 (PCA3) -RNA in situ in FFPE-prostate tissue using the RNAscope assay from ACDBio.
We will start the process of establishing in situ hybridization of DNA/mRNA in our laboratory as part of our “Learning from Deep Learning” project. We will further expand our sequential staining repertoire to include both mRNA and DNA in situ hybridization analysis. This will provide additional information on smaller chromosomal rearrangements and enable us to compare mRNA and protein expression directly in the tissue on a cell-by-cell basis. We will explore the possibilities of performing sequential CISH, using chromogens that are soluble in organic solvents. Alternatively, one step of CISH will be implemented as the last step of the sequential staining procedure.