m5C (5-methylcytosine) ELISA Kit

Epigenetics including DNA and RNA modifications have always been the hotspot field of life sciences in the post-genome era. While much attention has been focused on the classical epigenetic mark, 5-methylcytosine (m5C). Levels of m5C are variable in eukaryotes genomes, ranging from undetectable amounts to about 2% of total DNA in vertebrates. Moreover, m5C has been found in different RNA types, including mRNA, rRNA and tRNA of representative organisms from all kinds of species, and their distribution is species-specific. As reversible epigenetic modifications, the biological importance of m5C modifications of DNA/RNA affect the fate of the modified molecules and play important roles in various biological processes, that has been recognized widely. In humans, most m5C occurs at CpG sites, which are associated with embryonic development, diseases, and aging. Thus, determining the global changes in m5C levels is very necessary to fully understand the physiological and pathological status of biology, such as human disease.

The RayBio^® m5C ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of m5C in urine, serum, or plasma samples collected from humans, rats, or mice. The quantity of m5C is determined by a known m5C standard curve. The m5C kit has a high sensitivity of approximately 2.336 ng/ml. The performance was also verified to exhibit good characteristics in terms of linearity, stability, precision, and spiking & recovery. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples.

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Kit Components

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1-liter graduated cylinders
• Absorbent paper
• Distilled or deionized water
• SigmaPlot software (or other software which can perform four-parameter logistic regression models)
• Tubes to prepare standard or sample dilutions
• Orbital shaker
• Aluminum foil
• Plastic wrap

1. Prepare all reagents, samples and standards as instructed.
2. Add 50 μL standards or samples to appropriate well. Incubate for 10 minutes at room temperature.
3. Add 50 μL anti-m5C to appropriate well. Incubate 1 hour at room temperature. And wash 5 times.
4. Add 100 μL prepared HRP-Secondary antibody solution. Incubate 1 hour at room temperature. And wash 5 times.
5. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 2-10 minutes at room temperature.
6. Add 100 μL Stop Solution to each well. Read at 450 nm immediately.

The entire kit may be stored at -20°C to -80°C for up to 6 months from the date of shipment. For extended storage, it is recommended to store it at -80°C. Avoid repeated freeze-thaw cycles.