Pseudouridine, abbreviated by the Greek letter psi-Ψ, is an isomer of the nucleoside uridine in which the uracil is attached via a carbon–carbon instead of a nitrogen–carbon glycosidic bond. Ψ was first discovered in 1957. To date, Ψ has been recognized as an abundant post-transcriptionally modified nucleoside in the RNA of various organisms. Ψ is an irreversible modification found in transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA). The biosynthesis of Ψ is carried out via post-transcriptional modification of the substrate RNAs by a class of enzymes called pseudouridine synthases (PUS). Several studies have confirmed an important role for Ψ modification in different aspects of gene regulation which affect gene translation, splicing, telomere maintenance and gene expression regulation.

The RayBio^® Pseudouridine Dot Blot Assay Kit is a reliable, simple, and rapid method of quantifying the global Ψ RNA modification levels in RNA samples collected from humans, rats, mice and other species.

• Highly sensitive and specific
• Requires minimal sample preparation
• No specialty equipment required
• Suitable for high-throughput screening
• Compatible with various sample types and species
• ≥ 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.