N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNAs (mRNAs), affecting their structure, stability, and cellular interactions. In RNA, m6A plays a role in the initiation and progression of different types of human cancers. In DNA, m6A has been identified as an important epigenetic and epitranscriptomic marker in higher eukaryotes. Increasing evidence shows that dynamic m6A modification in genomic DNA is associated with brain functions, glioblastoma, embryogenesis, embryonic stem cell development, and cardiovascular disease. Determining the global changes in m6A levels is necessary to understand both the physiological and pathological states.

The global m6A level can be measured by dot blot or ELISA analysis using m6A-specific antibodies, mass spectrometry following chromatographic separation, or a high-throughput sequencing platform. Here, we describe an m6A dot blot kit that is a reliable, simple, and rapid method of quantifying the global m6A levels in RNA/DNA, 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 anti-m6A 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.