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SmFISH in chips: a microfluidic-based pipeline to quantify in situ gene expression in whole organisms.

ABSTRACT: Gene expression and genetic regulatory networks in multi-cellular organisms control complex physiological processes ranging from cellular differentiation to development to aging. Traditional methods to investigate gene expression relationships rely on using bulk, pooled-population assays (e.g. RNA-sequencing and RT-PCR) to compare gene expression levels in hypo- or hyper-morphic mutant animals (e.g. gain-of-function or knockout). This approach is limited, especially in complex gene networks, as these genetic mutations may affect the expressions of related genes in unforseen ways. In contrast, we developed a microfluidic-based pipeline to discover gene relationships in a single genetic background. The microfluidic device provides efficient reagent exchange and the ability to track individual animals. By automating a robust microfluidic reagent exchange strategy, we adapted and validated single molecule fluorescent in situ hybridization (smFISH) on-chip and combined this technology with live-imaging of fluorescent transcriptional reporters. Together, this multi-level information enabled us to quantify a gene expression relationship with single-animal resolution. While this microfluidic-based pipeline is optimized for live-imaging and smFISH C. elegans studies, the strategy is highly-adaptable to other biological models as well as combining other live and end-point biological assays, such as behavior-based toxicology screening and immunohistochemistry.

SUBMITTER: Wan J

PROVIDER: S-EPMC8146400 | biostudies-literature |

REPOSITORIES: biostudies-literature

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SmFISH in chips: a microfluidic-based pipeline to quantify in situ gene expression in whole organisms.

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