Measuring Pattern Separation in Hippocampus by in Situ Hybridization

Current protocols

2022 Aug 01

Eom, K;Lee, HR;
PMID: 35980141 | DOI: 10.1002/cpz1.522

Distinguishing different contexts is thought to involve a form of pattern separation that minimizes overlap between neural ensembles representing similar experiences. Theoretical models suggest that the dentate gyrus (DG) segregates cortical input patterns before relaying its discriminated output patterns to the CA3 hippocampal field. This suggests that the evaluation of neural ensembles in DG and CA3 could be an important means to investigate the process of pattern separation. In the past, measurement of entorhinal cortex (EC), DG, and CA3 ensembles was largely dependent upon in vivo electrophysiological recording, which is technically difficult. This protocol provides a method to instead measure pattern separation by a molecular method that provides direct spatial resolution at the cellular level.

Spatially Resolved and Highly Multiplexed Protein and RNA In Situ Detection by Combining CODEX With RNAscope In Situ Hybridization

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society

2022 Jul 16

Cheng, Y;Burrack, RK;Li, Q;
PMID: 35848523 | DOI: 10.1369/00221554221114174

Highly multiplexed protein and RNA in situ detection on a single tissue section concurrently is highly desirable for both basic and applied biomedical research. CO-detection by inDEXing (CODEX) is a new and powerful platform to visualize up to 60 protein biomarkers in situ, and RNAscope in situ hybridization (RNAscope) is a novel RNA detection system with high sensitivity and unprecedent specificity at a single-cell level. Nevertheless, to our knowledge, the combination of CODEX and RNAscope remained unreported until this study. Here, we report a simple and reproducible combination of CODEX and RNAscope. We also determined the cross-reactivities of CODEX anti-human antibodies to rhesus macaques, a widely used animal model of human disease.

Protocol for the use of signal amplification by exchange reaction-fluorescence in situ hybridization on adult formalin-fixed paraffin-embedded mouse lung tissue

STAR protocols

2023 Jun 09

Warren, R;Shaik, A;Teubner, L;Lyu, H;De Langhe, S;
PMID: 37302070 | DOI: 10.1016/j.xpro.2023.102353

Fluorescence in situ hybridization (FISH) is a useful tool for analyzing RNA expression, but difficulties arise with low-abundance RNA and in tissues that are formalin-fixed paraffin-embedded (FFPE) because reagents can be expensive. In this protocol, we adapt a previously designed FISH amplification protocol (SABER [signal amplification by exchange reaction]) for adult mouse FFPE lung sections by using probes that are extended and branched to amplify the signal. We combine FISH and immunostaining to identify cell-specific RNA. For complete details on the use and execution of this protocol, please refer to Kishi et al.1 and Lyu et al.2.

Multiomics Technologies Capture More Particulars, Reveal More Grandeur

Genetic Engineering & Biotechnology News

2023 Jun 01

LeMieux, J;
| DOI: 10.1089/gen.43.06.13

A patient's genome, Van Eyk noted, contains information about that patient's disease predispositions and drug responses. She added, however, that better information about disease risks and drug responses could be gleaned from the proteome. Although there are only so many protein-encoding genes, the intricacies of protein expression generate various kinds of proteomic information in abundance. According to Van Eyk, information about disease-induced modifications, isoforms, concentration changes, and chemical complexity can inform predictions of what will happen in the body, in the context of the body and the environment. She suggests that a proteomics approach—one that would involve monitoring of not just one protein at a time, but thousands—could generate valuable clinical insights.

Multiscale microscopy to decipher plant cell structure and dynamics

The New phytologist

2022 Dec 07

Cui, Y;Zhang, X;Li, X;Lin, J;
PMID: 36477856 | DOI: 10.1111/nph.18641

New imaging methodologies with high contrast and molecular specificity allow researchers to analyze dynamic processes in plant cells at multiple scales, from single protein and RNA molecules to organelles and cells, to whole organs and tissues. These techniques produce informative images and quantitative data on molecular dynamics to address questions that cannot be answered by conventional biochemical assays. Here, we review selected microscopy techniques, focusing on their basic principles and applications in plant science, discussing the pros and cons of each technique, and introducing methods for quantitative analysis. This review thus provides guidance for plant scientists in selecting the most appropriate techniques to decipher structures and dynamic processes at different levels, from protein dynamics to morphogenesis.

Gene expression data visualization tool on the o²S²PARC platform

F1000Research

2022 Nov 07

Ben Aribi, H;Ding, M;Kiran, A;
| DOI: 10.12688/f1000research.126840.1

Background: The identification of differentially expressed genes and their associated biological processes, molecular function, and cellular components are important for genetic diseases studies because they present potential biomarkers and therapeutic targets. Methods: In this study, we developed an o²S²PARC template representing an interactive pipeline for the gene expression data visualization and ontologies data analysis and visualization. To demonstrate the usefulness of the tool, we performed a case study on a publicly available dataset. Results: The tool enables users to identify the differentially expressed genes (DEGs) and visualize them in a volcano plot format. The ontologies associated with the DEGs are determined and visualized in barplots. Conclusions: The “Expression data visualization” template is publicly available on the o²S²PARC platform.

Efficient RNA and RNA-protein co-detection in 3D colonoids by whole-mount staining

STAR protocols

2022 Dec 16

Atanga, R;Parra, AS;In, JG;
PMID: 36313534 | DOI: 10.1016/j.xpro.2022.101775

Here, we describe a protocol to visualize RNA oligos and proteins independently or together using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence in human colonoids, expanding on previously published research. Whole-mount staining is used to preserve the colonoid structure and fix onto glass slides. We describe procedures for efficient plating, fixation, and preservation of the colonoids. This workflow can be adapted to 3D organoid models from other tissues or organisms. For complete details on the use and execution of this protocol, please refer to In et al. (2020).

Recent advances in single-cell subcellular sampling

Chemical communications (Cambridge, England)

2023 May 02

Sahota, A;Monteza Cabrejos, A;Kwan, Z;Paulose Nadappuram, B;Ivanov, AP;Edel, JB;
PMID: 37039236 | DOI: 10.1039/d3cc00573a

Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine.

Analysis of mitochondrial double-stranded RNAs in human cells

STAR Protocols

2023 Mar 01

Kim, S;Yoon, J;Lee, K;Kim, Y;
| DOI: 10.1016/j.xpro.2022.102007

Human mitochondrial genome is transcribed bidirectionally, generating long complementary RNAs that can form double-stranded RNAs (mt-dsRNAs). When released to the cytosol, these mt-dsRNAs can activate antiviral signaling. Here, we present a detailed protocol for the analysis of mt-dsRNA expression. The protocol provides three approaches that can complement one another in examining mt-dsRNAs. While the described protocol is optimized for human cells, this approach can be adapted for use in other animal cell lines and tissue samples. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).1

Double Immunohistochemical Staining on Formalin-Fixed Paraffin-Embedded Tissue Samples to Study Vascular Co-option

Methods in molecular biology (Clifton, N.J.)

2022 Sep 26

Annese, T;Errede, M;De Giorgis, M;Lorusso, L;Tamma, R;Ribatti, D;
PMID: 36161411 | DOI: 10.1007/978-1-0716-2703-7_8

Vascular co-option is a non-angiogenic mechanism whereby tumor growth and progression move on by hijacking the pre-existing and nonmalignant blood vessels and is employed by various tumors to grow and metastasize.The histopathological identification of co-opted blood vessels is complex, and no specific markers were defined, but it is critical to develop new and possibly more effective therapeutic strategies. Here, in glioblastoma, we show that the co-opted blood vessels can be identified, by double immunohistochemical staining, as weak CD31+ vessels with reduced P-gp expression and proliferation and surrounded by highly proliferating and P-gp- or S100A10-expressing tumor cells. Results can be quantified by the Aperio Colocalization algorithm, which is a valid and robust method to handle and investigate large data sets.

Targeting Alternative Splicing for Therapeutic Interventions

Methods in molecular biology (Clifton, N.J.)

2022 Jul 27

Centa, JL;Hastings, ML;
PMID: 35895256 | DOI: 10.1007/978-1-0716-2521-7_2

Targeting of pre-mRNA splicing has yielded a rich variety of strategies for altering gene expression as a treatment for disease. The search for therapeutics that can modulate splicing has been dominated by antisense oligonucleotides (ASOs) and small molecule compounds, with each platform achieving remarkably effective results in the clinic. The success of RNA-targeting drugs has led to the exploration of new strategies to expand the repertoire of this type of therapeutic. Here, we discuss some of the more common causes of faulty gene expression and provide examples of approaches that have been developed to target and correct these defects for therapeutic value.

Navigating the cellular landscape in tissue: Recent advances in defining the pathogenesis of human disease

Computational and Structural Biotechnology Journal

2022 Sep 15

Chen, H;Palendira, U;Feng, C;
| DOI: 10.1016/j.csbj.2022.09.005

Over the past decade, our understanding of human diseases has rapidly grown from the rise of single-cell spatial biology. While conventional tissue imaging has focused on visualizing morphological features, the development of multiplex tissue imaging from fluorescence-based methods to DNA- and mass cytometry-based methods has allowed visualization of over 60 markers on a single tissue section. The advancement of spatial biology with a single-cell resolution has enabled the visualization of cell-cell interactions and the tissue microenvironment, a crucial part to understanding the mechanisms underlying pathogenesis. Alongside the development of extensive marker panels which can distinguish distinct cell phenotypes, multiplex tissue imaging has facilitated the analysis of high dimensional data to identify novel biomarkers and therapeutic targets, while considering the spatial context of the cellular environment. This mini-review provides an overview of the recent advancements in multiplex imaging technologies and examines how these methods have been used in exploring pathogenesis and biomarker discovery in cancer, autoimmune and infectious diseases.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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