Isolation and Transcriptomic Profiling of Single Myofibers from Mice
[Abstract] Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of ...
Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity
[Abstract] Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for ...
Identification of RNA-binding Proteins
Authors: Kazuya Masuda and
Tadamitsu Kishimoto,
date: 09/05/2016,
view: 12443,
Q&A: 0
[Abstract] This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).
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Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
[Abstract] mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the ...
Cellular Translational Reporter Assay
Authors: Taishi Kimura and
Kiyoshi Takeda,
date: 03/20/2014,
view: 9974,
Q&A: 0
[Abstract] The method described here allows measuring the effect of exogenously introduced modifications to in vitro-transcribed mRNA on the translation in cells. Using cells derived from knockout mice and control littermates, this method enables to compare the results in the presence or absence of specific gene products. In our lab, we used this ...