SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping

Ghoddousi, Ramin Ali Marandi; Magalong, Valerie; Kamitakahara, Anna; Levitt, Pat

doi:10.1016/j.crmeth.2022.100316

Cited by 4 publications

(2 citation statements)

References 37 publications

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“…Registration was performed on sequential images from multiple cycles of immunofluorescence to adjust differences in alignment using the RNAscope HiPlex Image Registration Software 60 . DAPI channel images of each cycle were aligned using the DAPI channel image of the first cycle as a reference to obtain the registration transforms, which were then applied to the remaining channels of each cycle.…”

Section: Methodsmentioning

confidence: 99%

Spatial subcellular organelle networks in single cells

Venkatesan

Zhang

Marteau

et al. 2023

Sci Rep

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Organelles play important roles in human health and disease, such as maintaining homeostasis, regulating growth and aging, and generating energy. Organelle diversity in cells not only exists between cell types but also between individual cells. Therefore, studying the distribution of organelles at the single-cell level is important to understand cellular function. Mesenchymal stem cells are multipotent cells that have been explored as a therapeutic method for treating a variety of diseases. Studying how organelles are structured in these cells can answer questions about their characteristics and potential. Herein, rapid multiplexed immunofluorescence (RapMIF) was performed to understand the spatial organization of 10 organelle proteins and the interactions between them in the bone marrow (BM) and umbilical cord (UC) mesenchymal stem cells (MSCs). Spatial correlations, colocalization, clustering, statistical tests, texture, and morphological analyses were conducted at the single cell level, shedding light onto the interrelations between the organelles and comparisons of the two MSC subtypes. Such analytics toolsets indicated that UC MSCs exhibited higher organelle expression and spatially spread distribution of mitochondria accompanied by several other organelles compared to BM MSCs. This data-driven single-cell approach provided by rapid subcellular proteomic imaging enables personalized stem cell therapeutics.

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Section: Methodsmentioning

confidence: 99%

Spatial subcellular organelle networks in single cells

Venkatesan

Zhang

Marteau

et al. 2023

Sci Rep

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“…Following dual HiPlex RNAscope, immunohistochemistry, and imaging procedures, as detailed above, confocal image stacks were analyzed using the single-cell automated multiplex pipeline for RNA (SCAMPR) quantification and spatial mapping ( Ghoddousi et al, 2022 ). SCAMPR facilitates the analysis of highly multiplexed mRNA labeling at single-neuron resolution.…”

Section: Methodsmentioning

confidence: 99%

Ontogeny and Trophic Factor Sensitivity of Gastrointestinal Projecting Vagal Sensory Cell Types

McCoy

Kamitakahara

2023

eNeuro

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Vagal sensory neurons (VSNs) located in the nodose ganglion provide information, such as stomach stretch or the presence of ingested nutrients, to the caudal medulla via specialized cell types expressing unique marker genes. Here, we leverage VSN marker genes identified in adult mice to determine when specialized vagal subtypes arise developmentally and the trophic factors that shape their growth. Experiments to screen for trophic factor sensitivity revealed that brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) robustly stimulate neurite outgrowth from VSNsin vitro. Perinatally, BDNF was expressed by neurons of the nodose ganglion itself, while GDNF was expressed by intestinal smooth muscle cells. Thus, BDNF may support VSNs locally, whereas GDNF may act as a target-derived trophic factor supporting the growth of processes at distal innervation sites in the gut. Consistent with this, expression of the GDNF receptor was enriched in VSN cell types that project to the gastrointestinal tract. Lastly, mapping of genetic markers in the nodose ganglion demonstrates that defined vagal cell types begin to emerge as early as embryonic day 13, even as VSNs continue to grow to reach gastrointestinal targets. Despite the early onset of expression for some marker genes, expression patterns of many cell type markers appear immature in prenatal life and mature considerably by the end of the first postnatal week. Together, the data support location-specific roles for BDNF and GDNF in stimulating VSN growth, and a prolonged perinatal timeline for VSN maturation in male and female mice.Significance StatementSubpopulations of gastrointestinal-projecting vagal sensory neurons (VSNs) in the nodose ganglia convey satiety signals from the gut to the brain that regulate feeding behavior. Here, we examine gene expression in developing VSNs to better understand how they mature in perinatal life as they are exposed to ingestive stimuli. We demonstrate that thousands of genes are differentially expressed in the nodose ganglia of early postnatal mice compared to adults, indicating that VSNs continue to mature well after consumption of the first meal. The data further highlight the postnatal maturation of specific VSN subtypes and their sensitivity to trophic factors that stimulate growth and innervation of the gastrointestinal tract.

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Cassini: Streamlined and Scalable Method for in situ profiling of RNA and Protein

Lapique,

Kim,

Thom

et al. 2024

Preprint

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In the expanding field of spatial genomics, numerous methods have emerged to decode biomolecules in intact tissue. Advanced techniques based on combinatorial decoding can resolve thousands of features in a reasonable time but are often constrained by either the prohibitive costs associated with commercial platforms or the complexity of developing custom instruments. Alternatively, sequential detection methods, like single-molecule FISH, are easier to implement but offer limited multiplexing capability or signal amplification. Here, we introduce Cassini, a new approach for straightforward, cost-effective multiplexed measurements of mRNA and protein features simultaneously. Cassini leverages rolling circle amplification (RCA), known for its robust amplification and remarkable stability even after intense stripping, to serially detect each feature in under 20 minutes of total experimental time. The method also enables simultaneous immunostaining with either fluorophore-conjugated or DNA-barcoded antibodies, through an optimized immunostaining buffer. In a single overnight run, we show that Cassini can quantify 32 features (comprising both RNA and proteins) with sensitivity similar to state-of-the-art FISH techniques. We provide a comprehensive protocol alongside an online probe-design platform (cassini.me), aiming to enhance accessibility and user-friendliness. With our open-source solution, we aspire to empower researchers to uncover the nuances of spatial gene expression dynamics across diverse biological landscapes.

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SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping

Cited by 4 publications

References 37 publications

Spatial subcellular organelle networks in single cells

Spatial subcellular organelle networks in single cells

Ontogeny and Trophic Factor Sensitivity of Gastrointestinal Projecting Vagal Sensory Cell Types

Cassini: Streamlined and Scalable Method for in situ profiling of RNA and Protein

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