Mammalia

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    Protocols in Current Issue
    A Cell Culture Model that Mimics Physiological Tissue Oxygenation Using Oxygen-permeable Membranes
    Authors:  Chloe-Anne Martinez, Peter A. Cistulli and Kristina M. Cook, date: 09/20/2019, view: 565, Q&A: 0
    [Abstract] Dissolved oxygen and its availability to cells in culture is an overlooked variable which can have significant consequences on experimental research outcomes, including reproducibility. Oxygen sensing pathways play key roles in cell growth and behavior and pericellular oxygen levels should be controlled when establishing in vitro models. ...
    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: 1286, 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 ...
    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: 8718, 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 ...
    Detection and Analysis of Circular RNAs by RT-PCR
    Authors:  Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 8543, 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 ...
    Immunoprecipitation of Tri-methylated Capped RNA
    Authors:  Karen E. Hayes, Jamie A. Barr, Mingyi Xie, Joan A. Steitz and Ivan Martinez, date: 02/05/2018, view: 3919, Q&A: 0
    [Abstract] Cellular quiescence (also known as G0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27Kip1. Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an ...
    Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
    Authors:  Soufiane Aboulhouda, Rachael Di Santo, Gabriel Therizols and David Weinberg, date: 10/05/2017, view: 5672, Q&A: 0
    [Abstract] The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to ...
    A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism
    Author:  Hiroshi Arakawa, date: 05/20/2017, view: 5359, Q&A: 0
    [Abstract] While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in ...
    Polysome Fractionation to Analyze mRNA Distribution Profiles
    Authors:  Amaresh C. Panda, Jennifer L. Martindale and Myriam Gorospe, date: 02/05/2017, view: 14534, Q&A: 4
    [Abstract] Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that ...
    Polysome Profiling Analysis
    Authors:  Masahiro Morita, Tommy Alain, Ivan Topisirovic and Nahum Sonenberg, date: 07/20/2013, view: 32028, Q&A: 2
    [Abstract] Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.
    Detection and Cloning of Spliced Transcripts by RT-PCR
    Authors:  Bianca Hoffmann and Bastian Grewe, date: 04/20/2013, view: 9264, Q&A: 0
    [Abstract] Using a Reverse Transcriptase-PCR approach spliced transcripts can be converted to cDNA, amplified and cloned into an expression plasmid. Sequencing of the obtained cDNA allows identification of the splicing events that generated the detected RNA (Grewe et al., 2012).



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