Global DNA Methylation Assay–LINE-1

Synonyms

DNA methylation, 5-methylcytosine, methyl, 5-mC, mC, methylation, global DNA, DNA quantification

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Overview

Global DNA Methylation – LINE-1 Kit Overview

Active Motif's Global DNA Methylation – LINE-1 Kit is an ELISA-based assay for the detection and quantification of global DNA methylation (i.e., 5-methylcytosine (5-mC) levels) in human genomic DNA. The assay measures 5-mC levels of Long Interspersed Nucleotide Element 1 (LINE-1) repeats. LINE-1 repeats represent approximately 17-18% of the human genome and have a copy number of approximately half a million. It is well established in the literature that the methylation status of these LINE-1 repeats serve as a surrogate measure of global DNA methylation.

Because changes in genomic DNA methylation levels are observed in various human diseases, measuring global DNA methylation has become an essential research method. Aberrant LINE-1 methylation is linked to the onset and poor prognosis of various diseases, including cancer. The 96-well Global DNA Methylation – LINE-1 Kit is ideally suited for screening variances in 5-mC levels across different disease states, treatment conditions, clinical outcomes, or environmental factors.

In the Global DNA Methylation – LINE-1 Assay, the genomic DNA of interest is first enzymatically digested using the provided MseI enzyme to generate the appropriate fragments to hybridize to a biotinylated consensus sequence corresponding to human LINE-1 transposon. The LINE-1 probe is designed to assay a 290 bp region of the LINE-1 repeat element containing 88 cytosine residues, of which 12 are in a CpG context. Hybridized samples are immobilized to a 96-well plate and methylated cytosines are identified using a 5-methylcytosine antibody, HRP-conjugated secondary antibody and colorimetric detection reagents. Generating a standard curve using the included DNA standards with known LINE-1 methylation levels provides the relative level of 5-methylcytosine in each DNA sample.

Global DNA Methylation – LINE-1 Kit Highlights:

• Compatibility: Use with cell or tissue derived human genomic DNA.
• Sensitivity: Can detect a little as 0.5% methylation.
• Input: Can detect 5-mC from as little as 10 ng of input DNA.
• Quantitative: Included DNA standards contain known levels of LINE-1 methylation which can be used to determine % 5-mC.
• Quick and Easy: Simple ELISA-based assay can be finished in 5 hours.

Flow Chart of Global 5-hmC DNA ELISA Kit

(Click image to enlarge)

Contents

Global DNA Methylation – LINE-1 Kit Contents

The Global DNA Methylation – LINE-1 Kit contains sufficient reagents to perform 96 reactions. It is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes the following components:

• LINE-1 Probe (2.5 μM); Store at -20°C
• MseI enzyme (10 U/μl); Store at -20°C
• 10X MseI Reaction Buffer; Store at -20°C
• Methylated DNA Standard (10 ng/μl); Store at -20°C
• Non-methylated DNA Standard (10 ng/μl); Store at -20°C
• 5-Methylcytosine mAb; Store at -20°C
• Anti-mouse HRP-conj. secondary Ab; Store at 4°C
• Assay Buffer AM3; Store at 4°C
• 20X Wash Buffer; Store at 4°C
• 96-well Streptavidin plate; Store at 4°C
• Developing Solution; Store at 4°C
• Stop Solution; Store at RT
• 5X Hybridization Buffer; Store at RT
• 10X Buffer W; Store at RT
• Plate sealer; Store at RT
• 0.2 ml PCR stripwell tubes; Store at RT

Data

Global DNA Methylation – LINE-1 Kit Data

The Global DNA Methylation – LINE-1 Kit is an ELISA-based assay that is specially formulated for detection and quantitation of 5-methylcytosine (5-mC) levels of human genomic DNA. For added convenience, methylated and non-methylated DNA standards with known LINE-1 methylation levels are provided for generating a standard curve to determine relative levels of 5-methylcytosine of the DNA samples.

Figure 1: Global DNA Methylation – LINE-1 Assay shows changes in 5-mC levels resulting from treatment of HeLa cells with 5-azacytidine.

The Global DNA Methylation – LINE-1 Kit was used to assay 100 ng per well of MseI digested genomic DNA from HeLa cells or HeLa cells treated with 0.05 µM 5-azacytidine. Treatment of cells with 5-azacytidine, a known inhibitor of DNA methyltransferases (DNMTs), results in a decrease in global methylation as compared to untreated HeLa cells as expected. Using the provided standards and curve fitting software, the percent 5-methylcytosine for each DNA sample can be determined.

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Figure 2: Graphical representation of the standard curve generated with the Global DNA Methylation – LINE-1 Kit.

The % 5-methylcytosine (% 5-mC) represented by each DNA standard was plotted on the x-axis and the Net OD 450nm readings for both the standards (triangles) and samples (circles) were plotted on the y-axis. The best curve fit was determined using Readerfit software (https://Readerfit.com) and the % 5-mC values for the DNA samples were extrapolated from the curve. The dynamic range of the suggested standard curve can detect percent methylation in the range of 1% to 13%, although the assay has been shown to detect as little as 0.5% methylation.

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Figure 3: Global DNA Methylation – LINE-1 Assay shows changes in 5-mC levels in cfDNA with age.

Human serum samples from healthy “normal” adults <25 yo (N=6, mean: 21.83) and >60 yo (N=9, mean:65.89) were collected in serum separator tubes (SST). cfDNA was extracted using Active Motif’s Cell-Free DNA (cfDNA) Purification Kit (Cat no. 25503/25504) from 5ml of serum and quantified using a Qubit fluorometer. Following the Global DNA Methylation Line-1 Kit protocol, without MseI digestion, 25 ng of cfDNA per well was used per well. The % 5-mC for each sample was calculated as a percentage of detectable CpG residues in the LINE-1 probe region, using the methylated DNA standards included in the kit. All samples were done in triplicate. Boxplot shows 25%-75% (▢) , min-max (Ι), average (x), and median (-) % 5-mC.

FAQs

Global DNA Methylation – LINE-1 Kit FAQs

What is the LINE-1 probe?

The LINE-1 probe is a 5’ biotinylated oligo designed to hybridize to a 290bp region of the LINE-1 repeat element. This region contains 88 cytosine residues, of which 12 are in a CpG context.

Is this kit compatible with other species?

No, it can only be used with human samples because of the specificity of the LINE-1 probe. For other species, consider the Methylated DNA Immunopreciptation (MeDIP) Kit, which uses an antibody specific for methylated cytosines. This kit allows pulldown of methylated DNA fragments that can be detected by qPCR or NGS.

How much DNA do I need for this assay?

This kit is compatible with 10-200 ng of MseI-digested DNA per well. However, if the OD 450 nm readings fall outside the limits of detection for the assay (as determined by reading for the standard curve), we suggest performing a sample titration of the DNA to determine the appropriate quantity to use within the assay and performing the rest of the assay at the determined concentration. We suggest a starting concentration of 100 ng/well. 1 million cells contain approximately 6 micrograms of gDNA.

How long should I allow the assay to develop?

We recommend first looking at the lot-specific recommendation on the data sheet that comes with the kit. If you no longer have the data sheet, please contact technical service. However, this is only a guideline. It’s crucial to monitor the blue color development in the standard curve wells. The wells containing the highest concentrations of methylated DNA (STD 100) should turn medium to dark blue and the blank wells should be a faint blue color. Refer to this image as a guideline.

What DNA standards are included in the kit?

Both 100% methylated and 0% methylated DNA standards are included. These DNA standards are prepared from human Jurkat cell genomic DNA to provide researchers known quantities of LINE-1 methylation that can be used to approximate the level of 5-mC methylation in their DNA samples.

How can I quantify methylation in my samples?

For quantitative analysis, the % 5-mC can be calculated as either a percentage of CpG content or total cytosine. The included DNA standards can be used to generate a standard curve for either the % 5-mC associated with detectable CpG residues or the % 5-mC relative to the total cytosine content, both which have been determined for the standards. A best curve fit is determined and the % 5-mC values for the DNA samples are extrapolated from the curve.

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