m7G (N7-methylguanosine) Dot Blot Assay Kit

N7-methylguanosine (m7G), is a posttranscriptional modification of RNA that plays an essential role in RNA processing, metabolism, and function. m7G is the most ubiquitous mRNA cap modification and is also present in internal mRNA, microRNA, tRNA, and rRNA. m7G is positively charged and produced by the methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) complex. Emerging evidence suggests that the METTL1/WDR4 complex plays a role in brain development and disease pathogenesis. m7G has also been implicated in many types of tumorigeneses, including head, neck, lung, liver, colon, bladder, and teratoma. Investigations into m7G have found that this modification was crucial for tumor chemoresistance, vasculogenesis and prognosis. Determining the global changes in m7G levels is necessary to understand both the physiological and pathological states.

The global m7G level can be measured by dot blot or ELISA analysis using m7G-specific antibodies, mass spectrometry following chromatographic separation, or a high-throughput sequencing platform. Here, we describe an m7G dot blot kit that is a reliable, simple, and rapid method of quantifying the global m7G 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.