Mono-methylation of adenosine in the N1 position, N1- methyladensoine (m1A), is a reversible modification that has been found in tRNA, rRNA, mitochondrial RNA, and mRNA. Functionally, m1A dysregulation affects multiple cellular processes, including RNA structural stability, folding, interactions with proteins, cell viability, cell proliferation, and cell death. m1A tends to be conserved and has been implicated in many critical biological processes including development, disease pathogenesis, circadian rhythm, and embryonic stem cell fate transition, as well as age-related disorders such as Alzheimer’s disease.

The dynamic regulation of m1A in response to physiological stress and the dysregulated expression of m1A regulators have been correlated with tumorigenesis and cancer recurrence. High concentrations of m1A observed in urine and tissue specimens from patients with cancer, rheumatoid arthritis, and AIDS support m1A’s use as a functional biomarker. Current research is constantly uncovering new roles and mechanisms involving m1A. Determining the global changes in m1A levels is necessary to understand both the physiological and pathological states.

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