Epigenetics, including DNA and RNA modifications, have been the subject of extensive investigation during the post-genome era; with much attention focused on the classical epigenetic mark, 5-methylcytosine (m5C). Levels of m5C are variable in eukaryotic genomes, ranging from undetectable amounts to about 2% of total DNA. Across representative organisms from diverse species, m5C has been detected in various RNA types—including mRNA, rRNA, and tRNA—where its distribution proves to be species-specific. These m5C modifications of DNA/RNA are linked to important roles in various biological processes. In humans, most m5C occurs at CpG sites, which are associated with embryonic development, disease pathogenesis, and aging. Determining the global changes in m5C levels is necessary to understand both the physiological and pathological states.

The global m5C level can be measured by dot blot or ELISA analysis using m5C-specific antibodies, mass spectrometry following chromatographic separation, bisulfite sequencing, and methylated DNA immuno-precipitation sequencing. Here, we describe an m1A dot blot kit that is a reliable, simple, and rapid method of quantifying the global m1A 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-m5C 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.