A-to-I (Adenosine-to-Inosine) RNA editing, a widespread form of RNA modification, involves the conversion of adenosine to inosine in RNA molecules by the action of adenosine deaminases acting on RNA (ADARs). This modification has been thrust into the spotlight due to its substantial impact on biological and pathological processes. It achieves this by altering the structure and function of a variety of RNA molecules, including double-stranded RNA, transfer RNA, microRNA, and viral RNA. Mounting evidence indicates that A-to-I editing is pivotal in the pathogenesis of numerous diseases, ranging from cancer and autoimmune disorders to cardiovascular diseases, neurological and neurodegenerative conditions, as well as metabolic disorders. Furthermore, the editing of double-stranded RNA (dsRNA) through A-to-I modification also influences other RNA modifications, such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), thereby reciprocally affecting the development of these diseases.

The global A-to-I editing level can be measured by dot blot or ELISA analysis using Inosine-specific antibody, mass spectrometry following chromatographic separation, or a high-throughput sequencing platform. Here, we describe an A-to-I editing dot blot kit that is a reliable, simple, and rapid method of quantifying the global A-to-I editing levels in RNA isolated from cell, tissue, urine, serum, plasma, cell culture medium or other cell samples collected from humans, rats, mice and other species.

• Highly sensitive and specific
• Requires minimal sample preparation
• No specialty equipment required
• Suitable for high-throughput screening
• Compatible with various sample types and species
• ≥ 48 samples could be detected by 1 kit

• RNase-free tubes
• Heat blocker
• Plastic petri dish
• UV cross-linker
• Chemiluminescent Imaging system

1. Sample preparation: Depending on the type of sample (RNA, plasma, or serum), different methods of extraction, dilution, and denaturation are required.
2. Sample loading and crosslinking: The sample is spotted onto a nitrocellulose membrane, air dried, and crosslinked with UV light twice.
3. Methylene blue staining: One line of the spotted sample replicates is cut off and stained with methylene blue buffer to serve as an internal control.
4. Blocking and antibody incubation: The membrane is blocked with blocking buffer and then incubated with corresponding antibody and HRP-conjugated secondary antibody.
5. Detection and imaging: The membrane is treated with a mixture of detection buffers C and D, which catalyzes a color development reaction. The chemiluminescent signal is captured by an imaging system.

The entire kit may be stored at -20°C to -80°C for up to 12 months from the date of shipment. For extended storage, it is recommended to store it at -80°C. Avoid repeated freeze-thaw cycles.