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Using the Cecal Ligation and Puncture Model of Sepsis to Induce Rats to Multiple Organ Dysfunction

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RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities

Authors: Juan Pablo Tosar, Fabiana Gámbaro, Mauricio Castellano and Alfonso Cayota, date: 02/20/2021, view: 984, Q&A: 0

[Abstract]

Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed

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Advances in Proximity Ligation in situ Hybridization (PLISH)

Authors: Monica Nagendran, Adam M Andruska, Pehr B Harbury and Tushar J Desai, date: 11/05/2020, view: 1461, Q&A: 0

[Abstract] Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression ...

A CRISPR Competition Assay to Identify Cancer Genetic Dependencies

Authors: Vishruth Girish and Jason M. Sheltzer, date: 07/20/2020, view: 2917, Q&A: 0

[Abstract] The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can ...

Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis

Authors: Nigora Mukhamedova, Kevin Huynh, Hann Low, Peter J. Meikle and Dmitri Sviridov, date: 07/05/2020, view: 1770, Q&A: 0

[Abstract] Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and ...

Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells

Authors: Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 3410, Q&A: 0

[Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...

Protocol for in situ Proximity Ligation Assay (PLA) and Microscopy Analysis of Epidermal Growth Factor Receptor (EGFR) Homodimerization

Authors: Keiichi Ota and Taishi Harada, date: 11/05/2018, view: 4765, Q&A: 0

[Abstract] Oncogenic drivers play central roles in tumorigenesis as well as in tumor cell survival and proliferation. Mutations of the epidermal growth factor receptor gene (EGFR) that result in constitutive activation of the receptor tyrosine kinase have been identified as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). ...

Microarray, IPA and GSEA Analysis in Mice Models

Authors: Stephanie N. Oprescu, Katharine A. Horzmann, Feng Yue, Jennifer L. Freeman and Shihuan Kuang, date: 09/05/2018, view: 5944, Q&A: 0

[Abstract] This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety ...

Hypoxia Reporter Element Assay

Authors: Daelynn R Buelow and Sharyn D. D Baker, date: 08/05/2018, view: 3955, Q&A: 0

[Abstract] Hypoxia is a condition in which there is a decrease in oxygen supply to the cellular environment. Changes to cellular oxygen levels can lead to transcriptional changes of oxygen-regulated genes. Reporter assays are used to study gene expression alteration and modifications in response to environmental changes. Dual-reporter assays allow the ...

Detection and Analysis of Circular RNAs by RT-PCR

Authors: Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 13208, Q&A: 4

[Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...

An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins

Authors: Thomas J Macartney, Gopal P Sapkota and Luke J Fulcher, date: 11/20/2017, view: 6123, Q&A: 0

[Abstract] We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide ...

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