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Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient

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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: 527, 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 ...

In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay

Authors: G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 1001, Q&A: 0

[Abstract] The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification ...

Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation

Authors: Christopher Fields, Peike Sheng, Bret Miller, Tianqi Wei and Mingyi Xie, date: 04/20/2019, view: 1477, Q&A: 1

[Abstract] Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Additionally, we ...

Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos

Authors: Rachna Narayanan and Andrew C. Oates, date: 03/20/2019, view: 2276, Q&A: 0

[Abstract] In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to ...

Quantification of Queuosine Modification Levels in tRNA from Human Cells Using APB Gel and Northern Blot

Authors: Zaneta Matuszek and Tao Pan, date: 03/20/2019, view: 997, Q&A: 0

[Abstract] Queuosine (Q) is a hypermodified base in the wobble anticodon position of tRNAs coding for the amino acids Tyr, His, Asn, and Asp. tRNA Q-modification is introduced by a queuine tRNA-ribosyltransferase (TGT) that replaces the guanine base at G34 at these tRNAs with the modified base. tRNA Q-modification is widely distributed among prokaryotic and ...

Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells

Authors: Gal Haimovich and Jeffrey E. Gerst, date: 11/05/2018, view: 6006, Q&A: 1

[Abstract] Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017) that detects ...

mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells

Authors: Madara Ratnadiwakara and Minna-Liisa Änkö, date: 11/05/2018, view: 6007, Q&A: 0

[Abstract] Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs ...

Behavioral Evaluation of Seeking and Preference of Alcohol in Mice Subjected to Stress

Authors: Ana Canseco-Alba, Norman Schanz, Hiroki Ishiguro, Qing-Rong Liu and Emmanuel S. Onaivi, date: 10/20/2018, view: 1461, Q&A: 0

[Abstract] The alcohol preference model is one of the most widely used animal models relevant to alcoholism. Stressors increase alcohol consumption. Here we present a protocol for a rapid and useful tool to test alcohol preference and stress-induced alcohol consumption in mice. In this model, animals are given two bottles, one with a diluted solution of ...

Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy

Authors: Tan A Nguyen, Lachlan Whitehead and Ken C Pang, date: 06/20/2018, view: 2305, Q&A: 0

[Abstract] Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition ...

Dual-probe RNA FRET-FISH in Yeast

Authors: Gable M. Wadsworth, Rasesh Y. Parikh and Harold D. Kim, date: 06/05/2018, view: 2828, Q&A: 0

[Abstract] mRNA Fluorescence In Situ Hybridization (FISH) is a technique commonly used to profile the distribution of transcripts in cells. When combined with the common single molecule technique Fluorescence Resonance Energy Transfer (FRET), FISH can also be used to profile the co-expression of nearby sequences in the transcript to measure ...

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