scholarly journals Transcriptome-wide spatial RNA profiling maps the cellular architecture of the developing human neocortex

2021 ◽  
Author(s):  
Kenny Roberts ◽  
Alexander Aivazidis ◽  
Vitalii Kleshchevnikov ◽  
Tong Li ◽  
Robin Fropf ◽  
...  
Keyword(s):  
Gene Expression ◽  
New Technology ◽  
In Situ Hybridisation ◽  
Cell Types ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Human Neocortex ◽  
Formalin Fixed Tissue ◽  
Spatial Profiling

Spatial genomic technologies can map gene expression in tissues, but provide limited potential for transcriptome-wide discovery approaches and application to fixed tissue samples. Here, we introduce the GeoMX Whole Transcriptome Atlas (WTA), a new technology for transcriptome-wide spatial profiling of tissues with cellular resolution. WTA significantly expands the Digital Spatial Profiling approach to enable in situ hybridisation against 18,190 genes at high-throughput using a sequencing readout. We applied WTA to generate the first spatial transcriptomic map of the fetal human neocortex, validating transcriptome-wide spatial profiling on formalin-fixed tissue material and demonstrating the spatial enrichment of autism gene expression in deep cortical layers. To demonstrate the value of WTA for cell atlasing, we integrated single-cell RNA-sequencing (scRNA-seq) and WTA data to spatially map dozens of neural cell types and showed that WTA can be used to directly measure cell type specific transcriptomes in situ. Moreover, we developed computational tools for background correction of WTA data and accurate integration with scRNA-seq. Our results present WTA as a versatile transcriptome-wide discovery tool for cell atlasing and fixed tissue spatial transcriptomics.

scholarly journals Specific Detection of Lawsonia intracellularis in Porcine Proliferative Enteropathy Inferred from Fluorescent rRNA In Situ Hybridization

1998 ◽  
Vol 35 (2) ◽  
pp. 153-156 ◽  
Author(s):  
M. Boye ◽  
T. K. Jensen ◽  
K. Møller ◽  
T. D. Leser ◽  
S. E. Jorsal
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Lawsonia Intracellularis ◽  
Specific Detection ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Formalin Fixed Tissue ◽  
Proliferative Enteropathy ◽  
Formalin Fixed

Fluorescent in situ hybridization targeting 16S ribosomal RNA was used for specific detection of the obligate intracellular bacterium Lawsonia intracellularis in enterocytes from pigs affected by proliferative enteropathy. A specific oligonucleotide probe was designed and the specificity of the probe was determined by simultaneous comparison with indirect immunofluorescence assay for detection of L. intracellularis in formalin-fixed tissue samples from 15 pigs affected by porcine proliferative enteropathy. We used 10 tissue samples from pigs without proliferative mucosal changes as negative controls. The results showed that the oligonucleotide probe is specific for L. intracellularis and that fluorescent in situ hybridization targeting ribosomal RNA is a suitable and fast method for specific detection and histological recognition of L. intracellularis in formalin-fixed tissue.

scholarly journals Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0 identifies proteins enriched within inflammatory lesions in tissue sections and human clinical biopsies.

2021 ◽  
Author(s):  
BOCHENG YIN ◽  
Laura R Caggiano ◽  
Rung-Chi Li ◽  
Emily McGowan ◽  
Jeffery W. Holmes ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Biology ◽  
Cell Types ◽  
Specific Cell ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Novel Approach ◽  
Esophageal Tissue ◽  
Inflammatory Proteins

Tissue microenvironment properties like blood flow, extracellular matrix or proximity to immune infiltrate are important regulators of cell biology. However, methods to study regional protein expression in context of the native tissue environment are limited. To address this need we have developed a novel approach to visualize, purify and measure proteins in situ using Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0. We previously implemented AutoSTOMP to identify proteins localized to the vacuoles of obligate intracellular microbes at the 1-2 μm scale within infected host cells1. Here we report custom codes in SikuliX to specify regions of heterogeneity in a tissue section and then biotin tag and identify proteins belonging to specific cell types or structures within those regions. To enrich biotinylated targets from fixed tissue samples we developed a biochemical protocol compatible with LC-MS. These tools were applied to a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and b) characterize inflammatory proteins enriched in IgG4+ lesions in esophageal tissue. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools are limited.

scholarly journals Automated identification of the mouse brain’s spatial compartments from in situ sequencing data

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Gabriele Partel ◽  
Markus M. Hilscher ◽  
Giorgia Milli ◽  
Leslie Solorzano ◽  
Anna H. Klemm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Sequencing Data ◽  
Tissue Samples ◽  
Gene Markers ◽  
Smooth Transitions

Abstract Background Neuroanatomical compartments of the mouse brain are identified and outlined mainly based on manual annotations of samples using features related to tissue and cellular morphology, taking advantage of publicly available reference atlases. However, this task is challenging since sliced tissue sections are rarely perfectly parallel or angled with respect to sections in the reference atlas and organs from different individuals may vary in size and shape and requires manual annotation. With the advent of in situ sequencing technologies and automated approaches, it is now possible to profile the gene expression of targeted genes inside preserved tissue samples and thus spatially map biological processes across anatomical compartments. Results Here, we show how in situ sequencing data combined with dimensionality reduction and clustering can be used to identify spatial compartments that correspond to known anatomical compartments of the brain. We also visualize gradients in gene expression and sharp as well as smooth transitions between different compartments. We apply our method on mouse brain sections and show that a fully unsupervised approach can computationally define anatomical compartments, which are highly reproducible across individuals, using as few as 18 gene markers. We also show that morphological variation does not always follow gene expression, and different spatial compartments can be defined by various cell types with common morphological features but distinct gene expression profiles. Conclusion We show that spatial gene expression data can be used for unsupervised and unbiased annotations of mouse brain spatial compartments based only on molecular markers, without the need of subjective manual annotations based on tissue and cell morphology or matching reference atlases.

Expression of p53 during mouse embryogenesis

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 857-865 ◽  
Author(s):  
P. Schmid ◽  
A. Lorenz ◽  
H. Hameister ◽  
M. Montenarh
Keyword(s):  
Gene Expression ◽  
Cellular Differentiation ◽  
Cellular Proliferation ◽  
In Situ Hybridisation ◽  
Terminal Differentiation ◽  
Mouse Embryogenesis ◽  
Differentiated Cells ◽  
The Brain ◽  
P53 Mrna

By in situ hybridisation we have examined the expression of p53 during mouse embryogenesis from day 8.5 to day 18.5 post coitum (p.c.). High levels of p53 mRNA were detected in all cells of the day 8.5 p.c. and 10.5 p.c. mouse embryo. However, at later stages of development, expression became more pronounced during differentiation of specific tissues e.g. of the brain, liver, lung, thymus, intestine, salivary gland and kidney. In cells undergoing terminal differentiation, the level of p53 mRNA declined strongly. In the brain, hybridisation signals were also observed in postmitotic but not yet terminally differentiated cells. Therefore, gene expression of p53 does not appear to be linked with cellular proliferation in this organ. A proposed role for p53 in cellular differentiation is discussed.

Abstract 4281: Targeted or whole genome sequencing of formalin-fixed tissue samples

2014 ◽  
Author(s):  
Sarah Munchel ◽  
Yen Hoang ◽  
Yue Zhao ◽  
Joseph Cottrell ◽  
Brandy Klotzle ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed

Proteinase-activated receptors in ovine cervical function

2005 ◽  
Vol 17 (7) ◽  
pp. 693 ◽  
Author(s):  
Sharon E. Mitchell ◽  
John J. Robinson ◽  
Margaret E. King ◽  
Lynda M. Williams
Keyword(s):  
Gene Expression ◽  
In Situ Hybridisation ◽  
Prostaglandin F2α ◽  
Cervical Dilation ◽  
Proteinase Activated Receptors ◽  
Pregnant Ewe ◽  
High Level ◽  
And Function

In sheep, inflammation not only functions in cervical dilation at parturition, but also plays an important part in the non-pregnant ewe cervix, as demonstrated by the high level of expression of interleukin (IL)-8 at oestrus. Ewes artificially induced to ovulate have significantly lower levels of IL-8 gene expression at oestrus compared with natural oestrus, indicating an inhibition of inflammation and function, offering an explanation for the low rates of conception in vaginally inseminated synchronised ewes. To identify potential pro-inflammatory agents to combat the anti-inflammatory effects of hormonal synchronisation of oestrus, we have investigated the role of proteinase-activated receptor (PAR)-1 and PAR-2. To localise and measure the level of expression of these receptors, ovine-specific probes were derived for PAR-1 and PAR-2 and used for quantitative in situ hybridisation in the ovine cervix. Both PAR-1 and PAR-2 were expressed in the luminal epithelium of the cervix throughout the oestrous cycle, with expression being highest at oestrus. The gene expression of PAR-2 at oestrus was approximately 30% higher than that of PAR-1. Artificial synchronisation of oestrus by either an intravaginal progesterone sponge or prostaglandin F2α injections did not inhibit PAR-1 or PAR-2 expression at oestrus; rather, in the case of PAR-2, progesterone synchronisation increased it. Both synchronising procedures increased the expression of PAR-1 and PAR-2 during the luteal phase of the cycle. Therefore, agonists of PAR-1 and PAR-2 may be potentially useful pro-inflammatory agents countering the inhibition of inflammation by hormonal synchronisation.

scholarly journals Methods to Enhance Signal Using Isotopic In Situ Hybridization

2002 ◽  
Vol 50 (8) ◽  
pp. 1031-1037 ◽  
Author(s):  
Betty Ky ◽  
Paul J. Shughrue
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cell Types ◽  
Oxytocin Receptor ◽  
Specific Cell ◽  
Rat Hypothalamus ◽  
Low Levels ◽  
Tuberoinfundibular Peptide ◽  
Cryostat Sections

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

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