Nanjing Xinfan Bio: Improved Single Molecule Fluorescence in Situ Hybridization Technology

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Now, the research team led by Lucas Pelkmans of the Institute of Molecular Life Sciences at the University of Zurich has developed a new strategy based on branched DNA, allowing single-molecule fluorescence in situ hybridization (smFISH) to be expressed in high-throughput genetic analysis. .

Introduction to genetic analysis methods

To analyze gene expression, we have many choices: qPCR, chip and rna-seq. However, most expression analyses tend to focus on only one parameter: transcript abundance. They neither provide spatial information, ie where specific RNAs are located; nor can they determine differences in expression levels between cells because the study of cell populations makes this information lost.

In situ hybridization (ISH) this microscopic approach solves both of these problems. It utilizes labeled nucleic acid probes to determine the spatial location and abundance of DNA or RNA in tissues and cells. There are two forms of this method, fluorescence (FISH) and color development (CISH). Previously, FISH and CISH have been qualitative: either positive or negative. With the development of technology, quantitative versions have also been developed.

Single molecule fluorescence in situ hybridization (smFISH) is a novel gene expression analysis method that reports transcript abundance and spatial localization. But so far, smFISH has not been able to achieve genome-wide analysis. Now, researchers at the University of Zurich in Switzerland have reported a strategy for smFISH to plug in high-throughput wings. 1

The disadvantage of traditional single molecule fluorescence in situ hybridization

Lucas Pelkmans of the Institute of Molecular Life Sciences at the University of Zurich led the study. He explained that traditional smFISH has two major drawbacks that hinder its high-throughput applications. First, it produces a relatively weak signal with a low signal-to-noise ratio. Second, it cannot be extended because it requires high magnification, long imaging time, and the conditions of each transcript need to be optimized.

Traditional smFISH uses a series of short oligonucleotides to detect transcripts, each labeled with a fluorophore. Thus, there is a sufficient concentration of fluorescent molecules on the mRNA to produce a visible spot, but usually only at 60x or 100x magnification, using oil-immersed, large numerical aperture lenses.

Improvement of single molecule fluorescence in situ hybridization

The researchers developed their methods based on branched DNA. In this strategy, 15 pairs of oligonucleotide probes target a specific mRNA. These probes will serve as landing pads for primary preamplifier probes, as well as binding sites for some secondary amplifier probes, and finally a set of tertiary labeled probes.

The researchers explained that this is like planting 15 hybrid probes on a transcript. This method produces an amplified signal with a 100-fold increase in brightness and a 3-fold increase in signal-to-noise ratio, while imaging time is significantly reduced compared to conventional methods.

With this strong signal, the research team automates the experimental process. They established a branched DNA library of 928 human genes and tested them with cultured HeLa cells. In this process, they used a 384-well plate with one probe per well and the same hybridization and washing conditions. After hybridization, they imaged approximately 10,000 cells per well at 40x magnification and used an internally developed algorithm to extract transcript abundance and spatial features, such as whether the spots were close to the cell membrane, or the nucleus, concentrated or dispersed. They collected a total of 18 spatial variables and used computer clusters to assess their relationship to gene regulation.

The data suggest that genes that exhibit similar spatial characteristics and similar intercellular differences are also more likely to have similar functional roles.

Everyone agrees that transcription at the single cell level is full of noise. That is, two genetically identical cells in the cytoplasm may have completely different numbers of transcripts. However, protein levels do not necessarily reflect this transcript abundance. Researchers believe that the reliability of biological processes may be due to the regulation of mRNA subcellular localization, and their data support this view. Today, their goal is to test this hypothesis directly to determine whether "the spatial structure of the transcript buffers transcriptional noise."

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