RNA modifications are dynamic and reversible chemical modifications on substrate RNA that are regulated by specific modifying enzymes. They play important roles in the regulation of many biological processes in various diseases, including cancer, neurological disorders, cardiovascular diseases, metabolic diseases, genetic and developmental diseases, as well as immune disorders.

Advanced sequencing technologies have increasingly highlighted the significance of RNA modifications in human diseases which caught increasing attention in scientific research. Recent studies have mapped out the locations and abundance of key RNA modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), and N7-methylguanosine (m7G), primarily through antibody immunoprecipitation or chemical treatments coupled with next-generation sequencing. It is well known that through the “writing (Methyltransferase) - erasing (Demethyltransferase) -reading (Binding protein)” mechanisms, RNA modifications regulate the stability, translation, and localization of pivotal disease-related mRNAs to manipulate disease development. Further research into RNA modifications, their roles, inhibitors, and activators, is essential for disease diagnosis, treatment, and prognosis.

Here, we introduce a combined m1A, m7G, m6A, and m5C dot blot kit, which offers a reliable, simple, and rapid method of quantifying the 4 most common global RNA modification levels in RNA, urine, serum, plasma, or other nucleic samples collected from humans, rats, and mice.

• Highly sensitive and specific
• Requires minimal sample preparation
• No specialty equipment required
• Convenient for high-throughput screening in large-scale studies
• ≥ 48 samples could be detected by 1 kit

• RNase-free tubes
• Heat blocker
• 10 cm 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.