The MethylFlash™ Urine 5-Methylcytosine (5-mC) Quantification Kit (Fluorometric) is a complete set of optimized buffers and reagents to fluorometrically quantify 5-mC in urine using an inhibitory competitive immunoassay method. It is suitable for detecting total urinary 5-methylcytosine levels, resulting from whole body turnover or degradation of methylated DNA/RNA in urine from humans and animals. The urine samples can be in fresh or frozen form. The kit has the following advantages and features:

• Innovative fluorometric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 4 hours.
• 96 strip well microplate format makes the assay flexible: manual or high throughput analysis 
• Innovative kit composition enables background signals to be extremely low, which eliminates the need for plate blocking and allows the assay to be simple, accurate, reliable, and consistent.
• The level of 5-mC measured in human urine samples using this kit is comparable to that detected by HPLC method. 
• A novel assay principle allows high sensitivity to be achieved. The detection limit can be as low as 0.01 ng/assay well or 0.3 nM of 5-mC.
• Low input range of urine for each assay with a volume of 0.5 to 5 µl and an optimal volume of 1 µl.
• Optimized antibody and enhancer solutions allow high specificity to 5-mC, with no cross-reactivity to unmethylated cytosine. 
• Negative control and positive standard are included, which are suitable for quantification of 5-mC in free form and 5-mC contained in methylated DNA/RNA fragments from different urine samples.

Background Information
Nucleobase 5-methylcytosine (5-mC), a modified form of cytosine converted by cytosine methyltransferases, is widespread both in DNA and different cellular RNAs. The biological importance of DNA 5-mC methylation as a major epigenetic modification in phenotype and gene expression has been widely recognized. Recent data strongly suggests that RNA 5-mC methylation is also involved in the regulation of various biological processes including tRNA stability/recognition and mRNA translation. 

Urinary excretion of 5-mC including both 5-methyl-2-deoxycytidine and 5-methylcytidine is an indication of a whole body turnover or degradation of methylated DNA and RNA. The urinary 5-mC level can be altered by a change of the bodies’ turnover of methylated DNA/RNA or alteration of cellular DNA/RNA methylation status. A number of studies have indicated that 5-mC excreted in urine has the potential to act as a cancer biomarker, with an increased level arising from disease onset and progression. For example, an elevated level of urinary 5-mC was observed in lung cancer, breast cancer, and leukemia patients with active disease states. It was also shown that urinary 5-mC excretion is effected by Alzheimer’s disease or by radiation treatment. It is suggested that urinary 5-mC might be applicable as a biological marker for detecting some types of cancer and monitoring cancer progression after treatment with radiation or demethylation reagents. 

Chromatography-based techniques such as HPLC and TLC mass spectrometry are commonly used for detecting 5-mC in urine and although fairly acurate, are time consuming, less sensitive, and have low throughput with high costs. To address this problem, Epigentek offers the MethylFlash™ Urine 5-Methylcytosine (5-mC) Quantification Kit (Colorimetric) to quantify 5-mC or body turnover status of methylated DNA/RNA using urine samples.

Principle & Procedure
In this ELISA-like inhibitory competitive immunoassay assay, urine samples and 5-mC standard are first incubated with a 5-mC antibody solution and then transferred to the wells coated with methylated DNA (5-mC). The well is washed to remove any unbound reagents after incubation and then a detection antibody is added to generate a signal that can be measured fluorometrically by reading the fluorescent intensity in a microplate spectrophotometer. Because 5-mC in the urine sample inhibits the binding of 5-mC antibody to 5-mC coated on the well, higher concentrations of 5-mC in the urine sample lead to a reduced binding of the antibody to the 5-mC on the strip-well. Therefore the signal or fluorescent intensity measured from the well will be inversely proportional to the amount of 5-mC in the urine sample and the amount of 5-mC in the urine sample can be quantified by a comparison with a predetermined 5-mC standard.

Fig. 1. Schematic procedure for the MethylFlash™ Urine 5-Methylcytosine Quantification Kit (Fluorometric).

Fig. 2. 5-mC standard was added into the assay wells at different concentrations and then measured with the MethylFlash™ Urine 5-Methylcytosine Quantification Kit (Fluorometric)

Fig. 3. 5-mC level is quantified from different volumes of human urine using the MethylFlash™ Urine 5-Methylcytosine Quantification Kit (Fluorometric)