Single Molecule FISH Service

The field of genomics has undergone a significant transformation in the last decade with the advent of new technologies that allow us to explore the complexities of the genome in unprecedented detail. One such technology that has emerged as a powerful tool for studying gene expression and regulation is smFISH (single molecule fluorescence in situ hybridization).

smFISH is a technique that enables the visualization and quantification of individual RNA or DNA molecules with high sensitivity and specificity. It has revolutionized the way researchers study gene expression, DNA replication, and chromatin organization, among other things. In this article, we will take an in-depth look at this technology, its applications, advantages, and limitations.

Fig.1 Fluorescence confocal images of Cdh1 smFISH in mouse embryonic salivary gland, lung, and kidney. (Wang, 2018)

Applications of smFISH

smFISH has a wide range of applications in different research fields. Here are some of the most common applications:

1. smFISH can be used to study gene expression at the single-cell level, allowing cell-to-cell variability in gene expression patterns to be identified.
2. RNA localization: The smFISH can be used to study the spatial distribution of RNA molecules within cells, providing information about the subcellular localization of various RNA species.
3. smFISH can be used in conjunction with other techniques such as immunofluorescence to study RNA-protein interactions within cells.
4. smFISH can be used to detect and quantify RNA molecules in clinical samples, allowing for the development of diagnostic and prognostic biomarkers for diseases such as cancer.

Advantages of smFISH

• It enables visualization and quantification of specifically labeled RNA molecules at single molecule resolution.
• It supports single cell gene expression experiments through time and space.
• It allows accurate characterization of the spatio-temporal patterns of endogenous gene expression.
• It provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA.

Workflow of smFISH

smFISH works by using multiple short, fluorescently labeled oligonucleotide probes that are complementary to the target RNA and will be coupled with a fluorophore for imaging. These probes hybridize to the target RNA molecules in fixed cells, producing bright fluorescent spots that can be detected and counted by microscopy. This allows for both the quantification and localization of cytoplasmic and nuclear RNAs at a single cell level and single molecule resolution.

• Design smFISH probes that are complementary to the target RNA and will be coupled with a fluorophore for imaging.
• Prepare sample for imaging (can be live or fixed).
• Hybridize smFISH probes to target RNA in sample.
• Collect images with a suitable microscope.
• Analyze your data. Localized, quantify, and visualize molecules with software.

You can also perform smFISH at multiple time points during a time-course experiment to probe mRNA kinetics in space and time. You can use different spectral labels to distinguish sub-regions of an mRNA.

Fig.2 Workflow of smFISH. (Piskadlo, 2022)

FAQs

Q. How is smFISH different from traditional FISH?

A. Traditional FISH enables the detection of RNA molecules in a population of cells, whereas smFISH enables the detection of individual RNA molecules within cells.

Q. Is smFISH appropriate for all RNA or DNA molecules?

A. No, smFISH is designed to detect RNA molecules within cells.

Q. How many RNA species can be detected using smFISH?

A. smFISH can be used to detect multiple RNA species simultaneously using different fluorophores, depends on the availability of probes.

Q. What are some of the difficulties associated with using smFISH?

A. Some of the drawbacks of employing smFISH are the expense, technical expertise required, probe design, and the method's limited throughput.

Creative Biolabs' smFISH Service

Creative Biolabs proffers a cutting-edge molecular technique denominated smFISH service, which serves to detect and visualize distinct RNA molecules in individual cells. This potent and innovative method proffers the capability to visualize gene expression, chromosomal irregularities, and even the identification of specific microorganisms in a diverse range of research applications. Our squad of mavens employs state-of-the-art equipment and techniques to deliver dependable and accurate results that cater to the exigencies of our esteemed clients. Our smFISH service facilitates researchers to attain deeper insights into the foundational processes that underlie cell biology, disease, and development.

References

1. Wang, S.H.; Single molecule RNA FISH (smFISH) in whole-mount mouse embryonic organs. Current Protocols in Cell Biology. 2018, 83(1): e79.
2. Piskadlo, E.; et al. Design, labeling, and application of probes for RNA smFISH. Alternative Splicing: Methods and Protocols. 2022, 173-183.