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Optimization of Hybridization and Probe Design Parameters for Single Molecule RNA-FISH


There are many biological molecules in cells, including DNA, RNA and protein. Their expression levels can reflect the current statuses of cells and the changes in their expression patterns are associated with developmental as well as disease progression pathways. More importantly, these molecules can function as valuable biomarkers in the clinical and laboratory setting. To detect these molecules, a variety of methods have been developed. To assay large quantities and types of mRNA transcripts, RNA-sequencing is often used where dissociated cells or tissue are extracted in bulk and their contents are processed and sequenced via NGS platforms. Unfortunately, these tools only show expression changes on average. In addition, these bulk analyses methods do not provide important spatial information which can elucidate processes where expression patterns are closely related to its location, e.g. cell-cell communication. Therefore, tools which can spatially profile expression level of genes within the original context of a cell are continued to harbor interest in the clinical and research setting. For many years, fluorescence in situ hybridization (FISH) has been widely used for this application. In situ hybridization (ISH) is based on the specific binding between the target and its probes. The application of fluorescence probes helps to label the biomarker of interest and allows them to be observed and analyzed under a fluorescence microscope. Currently, many commercial RNA-FISH platforms exist but costs thousands of dollars to profile a small panel of genes for a small set of samples. Few pipelines tools which utilizes alternative cost-effective labeling strategies exist. The objective of this work is to develop a well-defined RNA-FISH platform which provides many guidelines for design of probe structure, blocking buffer, reagents, etc. We first constructed HEK293T cells that stably express mNeonGreen protein and validated the hybridization efficiency of the designed probes. Based on this cell system, the effects of primary probe structure, secondary probe structure, blocking buffer, primary probe concentration, and incubation time were studied. Based on their results and analyses, this pipeline for RNA-FISH experiments can be readily adopted and disseminated for use.

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