ArticleBio-protocol2025
An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues.
Article in Bio-protocol, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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4 authors.
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Abstract
Regulated IRE1-dependent decay (RIDD) is a critical cellular mechanism mediated by the endoplasmic reticulum (ER) stress sensor IRE1α, which cleaves a variety of RNA targets to regulate ER homeostasis. Current in vitro assays to study IRE1α activity largely rely on synthetic or in vitro transcribed RNA substrates, which may not fully replicate the physiological complexities of native RNA molecules. Here, we present a comprehensive protocol to assess IRE1α-dependent RNA cleavage activity using total RNA isolated directly from mouse tissues. This protocol provides a step-by-step guide for tissue collection, RNA isolation, an ex vivo RIDD assay, cDNA synthesis, and subsequent RT-PCR analysis of target mRNA cleavage products. Key reagents include active IRE1α protein, the RIDD-specific inhibitor 4μ8C, and target-specific primers for RIDD-regulated genes such as
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