An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues.

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 and . Quantitative assessment is achieved using agarose gel electrophoresis and imaging software. This methodology enables the study of IRE1α's RNA cleavage activity under conditions that closely mimic in vivo environments, providing a more physiologically relevant approach to understanding the role of RIDD in cellular and tissue-specific contexts. Key features • Uses total RNA from mouse tissues instead of synthetic RNA to better reflect in vivo conditions. • Includes RIDD-specific controls such as IRE1α inhibitor (4μ8C) and RNase A to confirm targeted RNA cleavage. • Combines agarose gel electrophoresis and ImageJ quantification for both qualitative and statistical validation. • Allows comparative studies of IRE1α activity across multiple mouse tissues in different biological contexts.