Among the spectrum of RNA modifications, N4-acetylcytidine (ac4C), referred to as RNA ac4C modification, is notable for its distinctive acetylation role. This modification is facilitated by the enzyme NAT10 and is consistently present in tRNA and rRNA. Notably, ac4C exhibits a dynamic distribution in mRNA, which is modulated by developmental stages and changes in NAT10 expression levels.

Recent scientific progress has started to address the challenges associated with ac4C modification, highlighting its natural variations in cancer, developmental processes, and immune responses. These variations are fundamental to protein synthesis and cellular metabolism. The ac4C modification strengthens the stability of RNA molecules, thereby prolonging their cellular lifespan and enhancing their functional capacity. This improvement ensures the accurate and efficient decoding of genetic information into proteins. Furthermore, this modification influences the RNA structure, which could potentially alter its interactions with ribosomes and the translation machinery. Such alterations have a substantial impact on the efficiency and accuracy of protein synthesis, which are crucial for the proper functioning of cells and their ability to adapt to environmental changes.

The global ac4C RNA modification level can be measured by dot blot or ELISA analysis using ac4C-specific antibody, mass spectrometry following chromatographic separation, or a high-throughput sequencing platform. Here, we describe an ac4C RNA modification dot blot kit that is a reliable, simple, and rapid method of quantifying the global ac4C RNA modification levels in RNA, urine, serum, plasma, or other nucleic 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.