Single-Cell Quantification of mRNA Expression in The Human Brain

Abstract

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis. Molecular characterisation of different cell-types in complex human tissues such as the brain is of crucial importance for both fundamental and applied research. The human brain is composed of potentially hundreds of different cell types displaying complex morphologies and often long, ramified and entwined processes, making its dissociation into single cells a difficult process. RNA-seq of immunopanned human brain cell types has been performed from fresh biopsies obtained from neurosurgical site 1 , but these samples are non-existent for neurodegenerative diseases; instead, neuroscientists normally access frozen or fixed post-mortem human brain samples from brain banks. However, single-cell preparation from snap frozen tissue is extremely difficult, thus making single-cell sorting virtually impossible. To overcome these limitations, several laboratories have performed single-nucleus RNA-seq. This method can be readily applied to frozen tissue samples and it has been shown to well recapitulate the cellular transcriptome 2,3. Nonetheless, this method has its own limitations, notably the loss of any compartmentalised cytoplasmic RNA 3. Single-cell or single-nucleus-based transcriptomics fail to capture spatial context, cell-to-cell relationships and circuitry structure. In situ hybridisation (ISH), which visualises specific mRNA molecules at a cellular level, is ideal to study gene expression in different cell types because it uses intact tissue sections, therefore maintaining tissue integrity, cell structure and morphology 4,5. Additionally, ISH has been successfully used for systematic analyses of RNA expression in the rodent brain (Allen Brain Atlas). Over the last decade, new and more sensitive ISH methods have been developed 4 , including single molecule RNA fluorescent in situ hybridization (smFISH) 6,7 , enabling quantification of RNA expression levels in specific cell types at single-cell resolution. Crucially, post-mortem human brain tissue presents higher rates of RNA degradation 8 , making bulk and single-cell or single-nuclei RNA analyses more complex. Alzheimer's disease (AD) is a devastating neurodegenerative disorder that accounts for two thirds of total dementia cases, and is characterised by progressive cognitive impairment and memory loss leading to difficulties in daily tasks 9. The AD brain displays extracellular...

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“…Once an ideal output could reflect the corresponding signals and separated individual cells, all pictures were analyzed using the module algorithm and a single H -score was obtained for each image, reflecting the overall target expression on a scale of 0 to 400. 34…”

dotdotdot: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues

“…While studies have begun to incorporate multiplex fluorescent approaches in postmortem human brain tissue, no consistent strategy for eliminating, masking, or subtracting lipofuscin autofluorescence has been described 3,5,[18][19][20][21][22] . Some studies have characterized lipofuscin autofluorescence based on size and intensity or custom filter cubes 3,4,21 , but these reports do not document how these approaches impact quantification of fluorescent signals from probe hybridization.…”

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“…2 ). 29 The proportion of GalR1 signal was then compared between SNAP25+/TH+ LC cells and SNAP25+/TH-cells surrounding the LC. Only 19.3% of GalR1 puncta were located in TH+ cells, while 80.7% of puncta were located in TH-cells ( Fig.…”

“…GalR1 mRNA expression in LC versus LC-adjacent neurons: To compare GalR1 mRNA expression in TH+ and TH-cells, LC sections were run with RNAscope probes for GalR1, TH, and Synaptosome Associated Protein 25 (SNAP25), a neuronal marker with an mRNA expression pattern that fills the cell soma. 29 The image channel corresponding to SNAP25 was used to segment individual cells and associated GalR1 puncta in 3-D. The TH channel was then overlaid and used to classify SNAP25+ cells as TH+ or TH-, and the proportion of GalR1 signal was calculated by cell type.…”

“…2). 29 The proportion of GalR1 signal was then compared between SNAP25+/TH+ LC cells and SNAP25+/TH-cells surrounding the LC.…”