5-methylcytosine (m5C), which involves the methylation of cytosine at the C5 position, stands as the most prevalent and reversible covalent modification of nucleotides in the human genome. This process of DNA methylation plays a pivotal role in regulating gene expression within healthy cells. Alterations in gene methylation patterns are not only linked to disease phenotypes but also serve as valuable biomarkers. Methylated DNA immunoprecipitation (MeDIP) is recognized as the gold standard for monitoring the status and mapping the precise locations of DNA modifications across specific genes and the entire genome.

Introducing the RayBio^® m5C (5-methylcytosine) DNA Enrichment and Quantification Kit (QMEDIP-M5C), a pioneering method designed to identify both the abundance and the enrichment region of m5C through a combination of MeDIP and real-time PCR assays. Enriched m5C DNA could also be used for genome-wide profiling of m5C. This assay entails a streamlined process where genomic DNA is first digested into fragments of approximately 200~500 nucleotides in length, followed by the application of a magnetic antibody specific to m5C. The enriched DNA is then released, purified, and eluted for subsequent qPCR analysis. The kit includes a magnetic non-immune IgG control and two pairs of primer controls to ensure accuracy and reliability. The QMEDIP-M5C kit offers a time-efficient and user-friendly procedure, capable of completing the entire process within 5 hours, with less than 1 hour of hands-on time required. It has been rigorously validated for the enrichment of m5C in human, mouse, and rat samples, making it a versatile tool for researchers in the field of epigenetics.

• Fast, streamlined procedure, finished in 5 hours
• Hands-on time is less than 1 hour
• Enriched RNA for transcriptome-wide profiling

• Fluorescence PCR instrument and compatible PCR plate
• Sterile nuclease-free pipette tips (barrier tips recommended) and microfuge tubes
• Vortex mixer
• Magnetic device
• DNA
• Primers for target gene of interest, with or without probe

1. Prepare all reagents, samples and standards as instructed.
2. DNA digestion.
3. m5C DNA immunoprecipitation (DIP).
4. qPCR assay for m5C-DIP.
5. Calculate the relative m5C enrichment.

The kit can be stored at -20°C for a period of 12 months prior to opening. After opening, the reagents are valid for at least 6 months if stored at -20°C. Avoid repeated freeze-thaw cycles.