What are the Differences Between DNA FISH and RNA FISH?

In situ hybridization (ISH) techniques initially developed by Joseph Gall and Mary Lou Pardue in the 1960s (Pardue and Gall 1969) and John et al. (1969) have proved to be powerful tools for determining the chromosomal location of hybridized nucleic acid. In the field of molecular biology, fluorescence in situ hybridization (FISH) techniques have revolutionized our understanding of the organization and function of genetic material within cells. FISH allows us to visualize and map specific DNA or RNA sequences in fixed cells or tissue samples, providing valuable insights into chromosomal abnormalities, gene expression patterns, and spatial organization of genetic material.

Principle of FISH

FISH techniques are based upon hybridization, with fluorescently labeled RNA or DNA probes that target and bind complementary DNA or RNA sequences in the sample of interest. The probes are made up of nucleotide sequences that are specific to the target sequences, enabling a high level of specificity in binding. Upon binding to its target sequence, the fluorescent label affixed to the probe generates a signal that is detectable via a fluorescence microscope.

Fig. 1 The principles of fluorescence in situ hybridization. (Shakoori AR, 2017)

What is DNA FISH?

DNA FISH is a powerful technique used to detect and localize specific DNA sequences within the genome. It has found wide applications in cytogenetics, cancer research, prenatal diagnosis, and gene mapping. In DNA FISH, the DNA probe is typically a short DNA fragment labeled with a fluorescent dye. Scientists use three different types of FISH probes, each of which has a different application.

Fig. 2 Sheep-specific gene probe (A), human telomere FISH probe (B), and human chromosome X paint probe (C).

• Locus-specific probes are designed to target specific loci or regions on a chromosome. They are useful for the detection of gene amplifications, deletions, translocations, and other structural rearrangements associated with genetic disorders or cancer. Locus-specific probes are valuable for identifying specific genetic markers or mutations within the genome.
• Alphoid or centromeric repeat probes target highly repetitive DNA sequences located at the centromeres of chromosomes. Centromeric FISH probes are essential for identifying numerical chromosomal abnormalities, such as aneuploidy (abnormal chromosome number), which are associated with conditions like Down syndrome (trisomy 21) or Turner syndrome (monosomy X).
• Whole chromosome probes are designed to hybridize to entire chromosomes. They are particularly valuable for assessing aneuploidy, polyploidy, and marker chromosomes. Whole chromosome probes can be used in prenatal screening for chromosomal abnormalities and in cancer cytogenetics to detect chromosomal rearrangements.

Applications of DNA FISH

• Gene mapping and localization - DNA FISH is widely used for gene mapping and localization. By designing probes that target specific genes or DNA sequences of interest, researchers can determine the precise location of these genetic elements within the genome. This information is valuable for studying gene structure, gain and loss, organization, and regulation.

Fig. 3 FISH mapping of LpGI gene. (Ansari HA, et al., 2017)

• Chromosome analysis and karyotyping - One of the primary applications of DNA FISH is in chromosome analysis and karyotyping. By using FISH probes specific to particular chromosomes or chromosomal regions, researchers can identify and visualize chromosomal abnormalities, such as translocations, deletions, duplications, and inversions.

Fig. 4 Karyotype and FISH analysis. (Yang R, et al., 2019)

• In medicine, FISH can be used for diagnosis, evaluation of prognosis, and evaluation of remission of a disease such as cancer. FISH can be used to detect diseased cells more easily than standard cytogenetic methods. It can be employed to identify a variety of genetic aberrations including gene gain or loss, translocations, and other changes in the genome.

Fig. 5 HER2 gene amplification testing by FISH for diagnosis of breast cancer. (Press MF, et al., 2016)

Fig. 6 Detection of double BCR/ABL fusion by FISH in chronic myeloid leukemia. (Dewald GW, et al., 1998)

What is RNA FISH & RNAscope in situ Hybridization?

While DNA FISH allows the detection of DNA sequences, RNA FISH is specifically designed to visualize and localize RNA molecules within cells. RNA FISH has become an essential tool in studying gene expression, RNA localization, and RNA-protein interactions. It allows researchers to directly observe the spatial and temporal distribution of RNA transcripts at the single-cell level, providing insights into gene expression patterns, RNA localization, and regulation within biological systems.

Launched in 2012, RNAscope is a novel RNA in situ hybridization (ISH) technology that detects target RNAs in tissues and cells. Its proprietary probe is designed to amplify target-specific signals but not background noise from non-specific hybridization. This technique enables the precise detection and spatial localization of RNA expression at single-molecule sensitivity, providing valuable insights into gene expression patterns within the context of intact biological samples.

Compared with traditional RNA in situ hybridization, the specific double Z probe design of RNAscope technology avoids the drawbacks of traditional long-stranded RNA probes and utilizes its own cascade amplification detection principle to detect the target RNAs with high sensitivity. Target RNAs with a specific sequence of greater than or equal to 300 bases can be used for the design of probes. As a result, RNAscope technology can be applied to almost all species, all tissues, and all genes.

• Sensitivity. Every three Z-shaped primer pairs are sufficient for the detection of a single RNA molecule. The design of 20 pairs of 2Z-primer pairs ensures strong enough RNA detection in cases where some RNA fragments cannot be effectively reached or where the RNA is partially degraded.
• Specificity. Z-pairs are designed to mask background noise. A single Z-primer probe targeting the binding of non-target sequences does not provide sufficient length for signal amplification of precursor sequence binding, thus preventing the amplification of non-specific signals and safeguarding specificity.
• Visualization and quantitative analysis at the single-molecule level. The 20x20x20x primer design and signal amplification principle allow a single RNA molecule to be visualized as a punctate signal under a standard microscope.

Fig. 7 The RNAscope principle. (Gross-Thebing T, et al., 2014)

Fig. 8 A schematic representation of Z probe-based RNAScope assay for lncRNA analysis. (Tripathi MK, et al., 2018)

Applications of RNA FISH & RNAscope in situ Hybridization

• Biomarker assay development
• Detection, characterization, and (co-) localization of miRNA/siRNA/lncRNA

Fig. 9 Detection of lncRNAs in mouse brain hippocampus.

Fig. 10 Evaluation of tissue PCA3 expression in prostate cancer by RNAscope. (Warrick JI, et al., 2014)

• Viral RNA detection
• Visualization of neuronal network activity and plasticity
• Assays of CAR-T and TCR-T cells

DNA FISH vs. RNA FISH

Although both DNA FISH and RNA FISH utilize similar principles, there are notable differences between the two techniques.

References

1. Shakoori AR. (2017). "Fluorescence In Situ Hybridization (FISH) and Its Applications." Chromosome Structure and Aberrations. 343-67.
2. Ansari HA, et al. (2017). "Fluorescence chromosome banding and FISH mapping in perennial ryegrass, Lolium perenne L." BMC Genomics. 17 (1): 977.
3. Yang R, et al. (2019). "Identification of chromosomal abnormalities and genomic features in near-triploidy/tetraploidy-acute leukemia by fluorescence in situ hybridization." Cancer Manag Res. 11: 1559-1567.
4. Gross-Thebing T, et al. (2014). "Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos." BMC Biol. 12: 55.
5. Wright JH, et al. (2021), Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry. J of Immun Methods. 492: 112955.
6. Wang F, et al. (2012). "RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues." J Mol Diagn. 14 (1): 22-9.
7. Press MF, et al. (2016). "HER2 Gene Amplification Testing by Fluorescent In Situ Hybridization (FISH): Comparison of the ASCO-College of American Pathologists Guidelines With FISH Scores Used for Enrollment in Breast Cancer International Research Group Clinical Trials." J Clin Oncol. 34 (29): 3518-3528.
8. Dewald GW, et al. (1998). "Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia." Blood. 91 (9): 3357-65.
9. Tripathi MK, et al. (2018). "Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues." Noncoding RNA. 4 (3): 20.
10. Warrick JI, et al. (2014). "Evaluation of tissue PCA3 expression in prostate cancer by RNA in situ hybridization--a correlative study with urine PCA3 and TMPRSS2-ERG." Mod Pathol. 27 (4): 609-20.