Global DNA Methylation
LINE-1 Kit(version A4)
Catalog No. 55017

Copyright 2014 Active Motif, Inc.

Revision History
Revision

Date

Description of Change

A2

Dec 2018

Clarified recommendations for colorimetric
reaction development time

A3

Sept 2020

Updated volume of LINE-1 Probe

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2020 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Traditional Methods to Study Global DNA Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Global DNA Methylation – LINE-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Global DNA Methylation – LINE-1 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section A. MseI Digestion of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section B. Standard Curve Preparation (Optional). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section C. DNA Sample Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section D. Binding to Streptavidin-coated Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section E. Binding of Primary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section F. Binding of Secondary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section G. Colorimetric Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section H. Analysis of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Appendix
Section I. . Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Section J. Sample-specific DNA Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Overview
The Global DNA Methylation – LINE-1 Kit is designed to detect methylation in Long Interspersed
Nucleotide Element 1 (LINE-1) repeat elements of human genomic DNA. LINE-1 methylation serves
as a surrogate for global DNA methylation levels and can be utilized to compare relative changes
in 5-methylcytosine levels across different sample types, treatment conditions, clinical outcomes,
dietary history or environmental backgrounds.
In the Global DNA Methylation – LINE-1 Kit, genomic DNA of interest is fragmented by enzymatic
digestion and hybridized to a biotinylated human LINE-1 consensus probe. Hybridized DNA isimmobilized onto a streptavidin-coated plate, while unbound DNA fragments are washed away. A
5-Methylcytosine antibody and a secondary antibody conjugated to horseradish peroxidase (HRP)
are used for detection of methylated fragments. The colorimetric readout is easily quantified byspectrophotometry using a microplate reader at 450nm.
The assay contains an optimized protocol and reagents necessary to perform DNA fragmentation,
hybridization, capture, and colorimetric detection of 5-methylcytosine. For added convenience,
methylated and non-methylated DNA standards containing known levels of LINE-1 methylation
are included in the kit. By generating a standard curve, the relative 5-methylcytosine levels ineach DNA sample can be determined.

product

formatcatalog no.

Global DNA Methylation – LINE-1 Kit

1 x 96 rxns

55017

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1Flow Chart of Process
MseI digested
genomic DNA

Biotinylated
LINE-1 probe

Hybridize DNA
and LINE-1 probe
in thermal cycler
70 min.

10

9

8

7

6

5

4

3

2

1

11

12

A
B
C
D
E
F

Immobilize to
streptavidincoated plate

G
H

1 hr.
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C

Wash and block
to reduce nonspecific binding
50 min.

D
E
F
G
H

1

12

11

10

9

8

7

6

5

4

3

2

A
B
C
D
E
F
G

Add 5-mC
antibody
1 hr.

H

21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F

Add anti-IgG
HRP conjugate

G
H

1 hr.
5

4

3

2

1

7

6

8

9

10

11

12

A
B
C
D
E
F
G
H

Add developing
and stop solution< 5 min.

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2

Introduction
DNA Methylation and Long Interspersed Nuclear Element 1 (LINE-1)
DNA methylation, which usually occurs at the fifth position of the cytosine pyrimidine ring of CpG
dinucleotides (5-mC) in mammals and other vertebrates, is an important epigenetic mechanism
regulating gene expression. Aberrant changes in DNA methylation have been linked to thedevelopment and progression of disease, including cardiovascular disease, lupus, cancer, birthdefects and neuropsychiatric disorders1-9. Consequently, DNA methylation patterns and/or levels
represent disease biomarkers with potential in both diagnostic and prognostic applications.
Repetitive elements comprise approximately 45% of the human genome and consist of either
tandem repeats of simple (satellite DNA) or complex sequences, or interspersed repeats derived
from non-autonomous or autonomous transposable elements10-14. It is estimated that more than
one-third of DNA methylation occurs in repetitive elements15-17. Literature indicates that analysisof the methylation status of high copy number repeat elements such as the long interspersed
nuclear element (LINE-1) can serve as a surrogate marker for global genomic DNA methylation18-20.
A comparison of the methylation status of the LINE-1 repeat elements correlates well with total
5-methylctyosine content when compared to high performance liquid chromatography (HPLC) or
combined bisulfite restriction analysis (COBRA) and pyrosequencing18,20. LINE-1 repeats represent
approximately 17-18% of the human genome, with a copy number estimated at roughly half a million21. LINE-1 hypomethylation is associated with elevated risk of several disease states including
cancer, stroke and heart disease and is considered a prognostic factor in gliomas22-28.
Using Active Motif’s Global DNA Methylation – LINE-1 assay, genomic DNA from different sample
types (e.g. normal and tumor), treatment conditions, clinical outcomes, dietary history or environmental backgrounds can be analyzed for differences in global DNA methylation levels. First,
genomic DNA of interest is enzymatically digested using the provided MseI enzyme to generate
the appropriate fragmentation 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.

References
1.
2.
3.
4.
5.
6.
7.

Esteller, M. et al. (2001) Cancer Res., 61(8): 3225-3229.
Jones, P.A. and Baylin, S.B. (2002) Nature Rev. Genet., 3(6): 415-428.
Chen, X. et al. (2010) Birth Defects Res. Part A. Clin. and Mol. Teratology 88(7): 575-581.
Feinberg, A.P. (2010) Virchows Archiv., 456(1): 13-21.
Wang, L. et al. (2010) American Journal of Clinical Nutrition, 91(5): 1359-1367.
Obermann-Bortst, S.A. et al. (2011) European Journal of Clinical Investigation, 41(2): 143150.
Szyf, M. (2011) Journal of Neurodevelopmental Disorders.
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8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.

Szyf, M. (2011) Toxicology Science, 120(2): 235-255.
Zhu, X. et al. (2011) International Journal of Dermatology, 50(6): 697-704.
Lander, E.S. et al. (2001) Nature, 409: 860-921.
Jordan, I.K. et al. (2003) Trends Genet., 19: 68-72.
Weiner, A.M. (2002) Curr. Opin. Cell. Biol., 14: 343-350.
Deininger, P.L. et al. (2003) Curr. Opin. Genet. Dev., 13: 651-658.
Prak, E.T. and Kazazian, H.H.J. (2000) Nature Rev. Genet., 1: 134-144.
Kochanek, S. et al. (1993) EMBO J., 12: 1141-1151.
Schmid, C.W. (1998) Nucleic Acids Res., 26: 4541-4550.
Bestor, T.H. (1998) Novartis Found. Symp., 214: 187-195.
Yang, A.S. et al. (2004) Nucleic Acids Res., 32(3): e38.
Weisenberger, D.J. et al. (2005) Nucleic Acids Res., 33(21): 6823-6836.
Lisanti, S. et al. (2013) PLoS One, 8(11): e79044.
Kazazian, H.H.J. (2000) Science, 289(5482): 1152-1153.
Ogino, S. et al. (2008) Journal of the National Cancer Institute, 100(23): 1734-1738.
Cho, Y.H. et al. (2010) Anticancer Research, 30(7): 2489-2496.
Irahara, N. et al. (2010) Journal of Molecular Diagnosis, 12(2): 177-183.
Wilhelm, C.S. et al. (2010) Clinical Cancer Research, 16(5): 1682-1689.
Baccarelli, A. et al. (2010) Epidemiology, 21(6): 819-828.
Kim, M. et al. (2010) PLoS One, 5(3): e9692.
Ohka, F. et al. (2011) PLoS One, 6(8): e23332.

Traditional Methods to Study DNA Methylation
To date, there are several methods used for DNA methylation analysis:
1. High performance liquid chromatography (HPLC): DNA can be digested into single
nucleotides which are separated according to size. Both cytosine and methylated cytosine
are quantified. Variants of this approach include tandem mas spectrometry (LC-MS/MS) as
well as two dimensional thin layer chromatography and high performance capillary electrophoresis. While these methods are highly quantitative, they require large amounts of DNA
and are not easily amenable to high throughput.
2. Bisulfite conversion: Bisulfite conversion consists of the treatment of genomic DNA withsodium bisulfite, leading to deamination of unmethylated cytosines into uracil. Converted
DNA is then PCR amplified and sequenced. Bisulfite-based techniques can be cumbersome,involving time- and labor-intensive chemical treatments that damage DNA and limit throughput. It is most readily used to analyze gene-specific sequences instead of global methylation.
3. Methylated DNA Immunoprecipitation (MeDIP): Methylated DNA Immunoprecipitation is
an immunocapture technique in which an antibody specific for methylated cytosines is used
to immunoprecipitate methylated genomic DNA fragments. Enriched DNA can be used foranalysis of the methylation status of a particular gene by PCR, or in combination with NGS.
MeDIP is prone to antibody bias and the technique does not consider total cytosine content.

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4Kit Performance and Benefits
The Global DNA Methylation – LINE-1 Kit is used to quantitate 5-methylcytosine levels in humangenomic DNA when comparing the samples to the provided DNA standards.
Range of detection: The dynamic range of the suggested standard curve can detect methylation
between 1% to 13%. The assay has been shown to detect as little as 0.5% methylation.
Input DNA can be used in the range of 10 ng - 200 ng per well. However, if the OD 450nm readings fall outside the limits of detection for the assay (as determined by the standard curve), wesuggest performing a sample titration of the DNA to determine the appropriate quantity to use
within the assay. Then perform the rest of the assay at the determined concentration. We recommend a starting concentration of 100 ng/well.
Cross-reactivity: The assay is specific to human LINE-1 repeat elements.
Assay time: 5 hours

Global DNA Methylation – LINE-1 Kit
Global DNA Methylation – LINE-1
2.5

OD 450nm

2
1.5
1
0.5
HeLa + Aza

DNA (100 ng/well)

HeLa

STD 100

STD 75

STD 50

STD 30STD 20

STD 10

STD 0

Blank

0

Figure 1: The Global DNA Methylation – LINE-1 assay compares 5-mC levels across treatment conditions.
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 standard standards and curve fitting software, the percent 5-methylcytosine for each DNA sample can be determined.

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5

Global DNA Methylation – LINE-1 Kit Components and Storage
Global DNA Methylation Kits are for research use only. Not for use in diagnostic procedures. Allcomponents are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

LINE-1 Probe (2.5 µM)

23 µl

-20°C

MseI enzyme (10 U/µl)

16 µl

-20°C

10X MseI Reaction Buffer

65 µl

-20°C

Methylated DNA Standard (10 ng/µl)

185 µl-20°C

Non-methylated DNA Standard (10 ng/µl)

270 µl-20°C

5-Methylcytosine mAb

110 µl-20°C

Anti-mouse HRP-conj. secondary Ab

450 µl4°C

Assay Buffer AM3

40 ml

4°C

20X Wash Buffer

25 ml

4°C

96-well Streptavidin plate

1 ea

4°C

Developing Solution

11 ml

4°C

Stop Solution

11 ml

RT

5X Hybridization Buffer

1 ml

RT

10X Buffer W

10 ml

RT

Plate sealer

2 ea

RT

0.2 ml PCR stripwell tubes

1 pk

RT

Additional materials required• Sample DNA• Orbital shaker (e.g. Multi-microplate Genie from Scientific Industries)• 1.5 or 2 ml microcentrifuge tubes• Microcentrifuge• Vortex• DNase-free sterile water• Nanodrop, Qubit or equivalent method to determine DNA concentration• (Optional) Graphing program with curve fitting capabilities

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6

Protocols
Buffer Preparation and Recommendations
MseI enzyme
The MseI enzyme is provided ready to use at 10 U/µl. Enough enzyme is provided to perform 32
DNA digestions as described in Step A of the protocol. Digested DNA is assayed in triplicate.
10X MseI Reaction Buffer
The reaction buffer is provided ready to use at a 10X concentration. Please follow the instructionsin the protocol for use.
LINE-1 Probe (See the Quick Chart for Preparing Buffers)
The LINE-1 Probe is provided at 2.5 µM. Prepare a master mix of probe diluted in Hybridization
Buffer for use with all assay samples, standards and blanks. Follow the recommendations in the
Quick Chart for Buffer Preparation to prepare only the amount of diluted probe needed for eachexperiment. Use 25 µl per well (0.5 pmol/well).
5X Hybridization Buffer (See the Quick Chart for Preparing Buffers)
The 5X Hybridization Buffer is provided ready to use. Please follow the recommendations in the
Quick Chart for Buffer Preparation for use.
1X Buffer W (See the Quick Chart for Preparing Buffers)
Prepare the amount of 1X Buffer W required for the assay as follows: For every 10 ml of 1X Buffer
W required, dilute 1 ml 10X Buffer W with 9 ml sterile water.
5-Methylcytosine mAb (See the Quick Chart for Preparing Buffers)
Prepare a 1:100 dilution of 5-methylcytosine monoclonal antibody into Assay Buffer AM3. Follow
the recommendations in the Quick Chart for Buffer Preparation to prepare only the amount ofantibody needed for each experiment. Use 100 µl per well.
Anti-mouse HRP-conj. secondary antibody (See the Quick Chart for Preparing Buffers)
Prepare a 1:25 dilution of anti-mouse HRP-conj. secondary antibody into Assay Buffer AM3. Follow the recommendations in the Quick Chart for Buffer Preparation to prepare only the amount ofantibody needed for each experiment. Use 100 µl per well.
1X Wash Buffer (See the Quick Chart for Preparing Buffers)
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of 1X
Wash Buffer required, dilute 5 ml 20X Wash Buffer with 95 ml sterile water. Mix gently to avoid
foaming. The 1X Wash Buffer may be stored at 4°C for one week. The Tween 20 contained in the
20X Wash Buffer may form visible aggregates, therefore, homogenize the buffer by incubating at
50°C for 2 minutes and mixing prior to use.
Developing Solution (See the Quick Chart for Preparing Buffers)
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense light during storage. The Developing Solution may develop a yellow hue over time. This does not affectwww.activemotif.com

7

product performance. However, a blue color present in the Developing Solution indicates thatit has been contaminated and must be discarded. Prior to use, place the Developing Solution atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required for theassay into a secondary container before aliquoting into the wells. After use, discard remaining
Developing Solution.
Stop Solution (See the Quick Chart for Preparing Buffers)
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary container. After use, discard remaining Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.
Methylated and Non-methylated DNA Standards
Methylated and Non-methylated DNA Standards are provided ready to use at a concentration of
10 ng/µl. Both standards are derived from human Jurkat genomic DNA. The methylated standard
has been in vitro methylated with M.SssI, then digested with MseI to produce the appropriate
fragmentation. The Non-methylated DNA Standard was whole genome amplified to remove
endogenous methylation. DNA Standards can be used as assay controls, or can be mixed in varying proportions to create a standard curve for quantitation of percent methylated cytosine (seeSection B of the protocol). Enough DNA is provided to perform two standard curves in triplicate.
Please follow the instructions in the protocol for use.
The provided DNA standards are suitable for relative quantitation, but do not account for copy
number variances that may exist in different sample types. If copy number variation is a concern,
please follow the recommendations in Appendix Section J to prepare Methylated and Non-methylated DNA standards for your specific DNA sample.

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8Quick Chart for Preparing Buffers
Reagents to prepare

Components

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

LINE-1 Probe Solution

LINE-1 Probe
5X Hybridization Buffer
Distilled water
TOTAL REQUIRED

0.2 µl10 µl
14.8 µl
25 µl

1.8 µl90 µl133.2 µl
225 µl10.8 µl
540 µl800 µl1.35 ml21.6 µl1.08 ml
1.6 ml
2.7 ml1X Buffer W

Distilled water
10X Buffer W
TOTAL REQUIRED

720 µl80 µl800 µl7.2 ml0.8 ml8 ml

40.5 ml
4.5 ml
45 ml

81 ml
9 ml
90 ml

5-Methylcytosine Ab

5-Methylcytosine Ab
Assay Buffer AM3
TOTAL REQUIRED

1 µl
99 µl
100 µl9 µl891 µl900 µl54 µl5.35 ml
5.4 ml108 µl10.7 ml
10.8 ml

HRP-conjugatedanti-mouse Ab

HRP-conjugated anti-mouse IgG
Assay Buffer AM3
TOTAL REQUIRED

4 µl
96 µl
100 µl36 µl864 µl900 µl

216 µl5.2 ml5.4 ml432 µl10.4 ml
10.8 ml

1X Wash Buffer

Distilled water
20X Wash Buffer
TOTAL REQUIRED

1.9 ml100 µl2 ml

17.1 ml
0.9 ml
18 ml

95 ml
5 ml
100 ml

190 ml
10 ml
200 mlDeveloping Solution

TOTAL REQUIRED

100 µl900 µl5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

100 µl900 µl5.4 ml10.8 mlwww.activemotif.com

9Assay Protocol
Read the entire protocol before use.
Prior to starting the Global DNA Methylation – LINE-1 Kit, genomic DNA is fragmented using
restriction digestion with the provided MseI enzyme. This enzymatic digestion will generate theappropriate fragments in the LINE-1 repeat elements of the genomic DNA to enable hybridization
to the assay’s LINE-1 probe.

Section A: MseI Digestion of Genomic DNA
We recommend preparing high-quality genomic DNA using a commercially available kit or astandard established protocol and determining DNA concentration by UV spectrophotometry or
fluorescence-based quantitation. The LINE-1 Global DNA Methylation assay can be performed on
approximately 100 ng MseI fragmented genomic DNA per well. We suggest using 1 µg genomic
DNA per sample type for digestion and then performing the assay in triplicate.
1.

In microcentrifuge tubes, set up a restriction digest for each DNA sample to be tested.
Also, prepare a mock digestion reaction that will be used as a blank for DNA quantification
following the digestion reaction. A small loss of DNA is anticipated as part of the digestion
process, therefore quantification MUST be performed following MseI digestion to accurately
determine the DNA quantity for use in the assay. A single mock digestion reaction can beused as a blank for all the DNA digestions performed on the same day.
Reagents

DNA
SampleMock
Digestion

Genomic DNA (1 µg)______ µl

0 µl

10X Reaction Buffer

2 µl

2 µl

MseI enzyme (10 U/µl)

0.5 µl

0.5 µlSterile water

Up to 20 µl

17.5 µl

Total Volume

20 µl

20 µl

2.

Mix well by pipetting and incubate at 37°C for 4 hours or overnight.

3.

Heat-inactivate MseI by incubating the reaction mixture at 65°C for 20 minutes. Quick spintubes to collect any condensate.

4.

Measure the DNA concentration of each sample using the mock digestion as a blank. We
suggest using 1 µl of the digestion reaction for quantification. The Global DNA Methylation
assay is very sensitive to DNA concentration so DNA MUST be quantified following MseI
digestion. Failure to properly quantify the DNA following digestion will lead to variability
in the assay results. Make sure your samples fall within the range of detection for your spectrophotometer or use a more sensitive fluorescence-based quantitation.

5.

If the assay will not be performed immediately, the digested DNA can be stored at -20°C.

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10Section B: Standard Curve Preparation (Optional)
The provided Methylated and Non-methylated DNA Standards can be used as assay controls for
non-quantitative analysis, or can be mixed in varying proportions to create a standard curve forquantitation of percent methylated cytosine in each sample. For non-quantitative assay controls
we recommend using: Blank, STD 0 and STD 100 as shown in the table below. To create a quantitative standard curve, prepare the entire range of standards.
1.

In microcentrifuge tubes, prepare a mixture of Methylated and Non-methylated DNA standards according to the following table. Volumes listed below are sufficient to prepare one
standard curve in triplicate.
Standard
Name

MethylatedStandard (ng)Non-Meth.
Standard (ng)VolumeMethylatedStandard

Volume NonMeth.
Standard

Volume
Water

BLANK

0

0

0 µl

0 µl

80 µl

STD 0

0

100

0 µl

32 µl

48 µl

STD 101090

3.2 µl28.8 µl

48 µl

STD 202080

6.4 µl25.6 µl

48 µl

STD 303070

9.6 µl22.4 µl

48 µl

STD 50

50

50

16 µl

16 µl

48 µl

STD 75

75

25

24 µl

8 µl

48 µl

STD 100

100

0

32 µl

0 µl

48 µl

2.

Vortex to mix thoroughly. Quick spin to collect liquid.

3.

Use 25 µl of each Standard per well.

Section C: DNA Sample Hybridization
1.

Determine the appropriate number of 0.2 ml stripwell PCR tubes required for testing
samples, standards and blanks in triplicate. Label each tube.

2.

Add 25 µl LINE-1 Probe Solution prepared on page 9 to all PCR tubes being used.

3.

Dilute the MseI digested DNA samples to a concentration of 4 ng/µl in sterile water. Mix wellby vortexing. (e.g. To prepare triplicate reactions, dilute 320 ng of DNA in sterile water for a
final volume of 80 µl. Use 25 µl per reaction.)

4.

Add DNA to the appropriately labeled PCR tube. The final volume is 50 µl per tube.
Blanks: Add 25 µl Blank per well (no DNA wells)
DNA Standards: Add 25 µl of each DNA Standard per well (100 ng DNA/well)
MseI-digested Samples: Add 25 µl of each DNA Sample per well (100 ng DNA/well)5.

Cap tubes and briefly vortex to mix. Collect liquid to bottom of tubes.

6.

Place samples in a thermal cycler and incubate at 98°C for 10 minutes, then quickly ramp to
68°C for 1 hour followed by a quick ramp to 25°C.
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11Section D: Binding to Streptavidin-coated Plate
Remove the appropriate number of microwell strips required for testing. Store the unused strips
in the aluminum pouch at 4°C. If less than 8 wells in a strip need to be used, cover the unused
wells with a portion of the plate sealer while you perform the assay. The unused wells are stableat room temperature for the duration of the assay if kept dry. Once the assay is finished, unused
strips should be returned to the aluminum pouch and stored at 4°C for a separate assay. Use thestrip holder while performing the assay. An example plate layout is shown below.
Prepare buffers as described in the section Buffer Preparation and Recommendations. Multichannel pipettor reservoirs may be used for dispensing the Buffer W, 1X Wash Buffer, Developing
Solution and Stop Solution into the wells being used.
DNA Standards

MseI-digested Samples

1

2

3

4

5

6

A

Blank

Blank

Blank

Sample 1

Sample 1

Sample 1

B

STD 0

STD 0

STD 0

Sample 2 Sample 2 Sample 2C

STD 10

STD 10

STD 10Sample 3 Sample 3 Sample 3

D

STD 20STD 20STD 20Sample 4 Sample 4 Sample 4

E

STD 30

STD 30

STD 30Sample 5 Sample 5 Sample 5F

STD 50STD 50STD 50Sample 6 Sample 6 Sample 6

G

STD 75

STD 75

STD 75

Sample 7

H

STD 100

STD 100

STD 100

Sample 8 Sample 8 Sample 8

Sample 7

Sample 7

1.

Collect any condensate to the bottom of the PCR tubes. Transfer the contents of each PCR
tube to a separate well of the streptavidin-coated assay plate. Use the provided adhesive
cover to seal the plate. Incubate for 1 hour at room temperature with mild agitation (100300 rpm on an orbital plate shaker). Keep any unused wells covered during the remaining
steps in order to preserve those wells for future assays.

2.

Remove the plate sealer and discard the unbound DNA by quickly inverting the plate over aliquid waste receptacle. Then, tap the inverted plate 3 times on absorbent paper towels.

3.

Add 200 µl 1X Buffer W to each well for a quick rinse and immediately discard solution as
described in Step 2. Repeat for a second rinse.

4.

Wash each well 2 times with 200 µl 1X Buffer W and incubate each wash for 10 minutes withmild agitation.

5.

Add 200 µl Assay Buffer AM3 to each well. Cover the plate with the adhesive sealer andincubate at room temperature for 30 minutes with mild agitation.

6.

During this incubation, prepare the 5-methylcytosine antibody dilution as described in thesection Buffer Preparation and Recommendations.

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12Section E: Binding of Primary Antibody
1.

Remove the plate sealer and discard the blocking buffer by quickly inverting the plate overa liquid waste receptacle, then tap the inverted plate 3 times on absorbent paper towels.

2.

Add 100 µl of diluted 5-Methylcytosine antibody to each well being used, including blankwells. Cover the plate and incubate for 1 hour at room temperature with mild agitation.

3.

During this incubation, prepare the anti-mouse HRP antibody dilution as described in thesection Buffer Preparation and Recommendations.

4.

After the incubation, rinse the wells 3 times with 200 µl 1X Wash Buffer.

Section F: Binding of Secondary Antibody1.

Add 100 µl of diluted HRP-conjugated anti-mouse antibody to each well being used, including blank wells. Cover the plate and incubate for 1 hour at room temperature with mild
agitation.

2.

During this incubation, place the Developing and Stop Solutions at room temperature.

3.

After the incubation, rinse the wells 3 times with 200 µl of 1X Wash Buffer.

Section G: Colorimetric Reaction
1.

Remove as much of the final wash as possible by blotting the plate on paper towels.

2.

Add 100 µl of room temperature Developing Solution to all wells being used.

3.

Incubate for the amount of developing time specified on the Certificate of Analysis at room
temperature, protected from direct light. Please read the Certificate of Analysis suppliedwith this kit for optimal development time associated with this lot number. Monitor the blue
color development in the standard curve wells containing the highest concentrations ofmethylated DNA (STD 100) until they turn medium to dark blue. The Blank wells should be a
faint blue color. Do not overdevelop.

4.

Add 100 µl of Stop Solution to all wells. In presence of the acid, the blue color turns yellow.

5.

Read absorbance on a spectrophotometer within 5 minutes at 450nm with an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.
Reading the reference wavelength is optional. Most microtiter plate readers are equippedto perform dual wavelength analysis and with the appropriate software, will automaticallysubtract the reference wavelength absorbance from the test wavelength absorbance. If
your plate reader does not have this capability, you may read the plate twice, once at 450
nm and once at 655 nm then manually subtract the 655 nm OD from the 450 nm OD values.

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13Section H: Analysis of Results
1.

Average the triplicate readings for all Blank, Standard and Sample wells. Then, subtract the
averaged Blank OD 450nm values from the average Standard and the averaged Sample
values. These are the Net readings.

2.

For non-quantitative analysis, OD 450nm values for samples can be compared to the assay
controls to determine relative levels of LINE-1 DNA methylation. The Blank wells represent
the baseline signal from the assay reagents in the absence of DNA. The STD 0 (nonmethylated DNA only) value displays the background signal of the assay in the presence ofunmethylated DNA samples, while the STD 100 (methylated DNA only) represents a sample
with a high degree of DNA methylation.

3.

For quantitative analysis, the % 5-mC associated with each sample can be calculated two
different ways. The first method determines % 5-mC based on the CpG content. The second
method determines the % 5-mC of CpG residues as it relates to total cytosine content. Determine the method of quantitation that is most appropriate for your experimental analysis.
Method 1: Determines the % 5-mC associated withthe detectable CpG residues.

Standard
Name

The 290 bp LINE-1 MseI fragments that hybridize to
the LINE-1 probe contain 10 detectable CpG residues.
The provided DNA standards are either 100% methylated or 0% methylated for all 10 CpGs. Therefore,
the standard curve generated by mixing the DNA
standards together quantitates methylation in therange of 0% to 100% 5-mC for the CpG content of thesamples. See the values to the right for the specific% 5-mC for each DNA Standard.

STD 0

0%

STD 1010%

STD 2020%

STD 3030%

STD 5050%

STD 7575%

STD 100

100%

Method 2: Determines the % 5-mC measured relative to the total cytosine content.

Standard
Name% 5-mCThe 290 bp LINE-1 MseI fragments that hybridize to
the LINE-1 probe contains 10 detectable CpG residues
out of a total of 76 cytosines. The provided DNA standards are either 100% methylated or 0% methylated
for all 10 CpGs. By dividing the methylated CpG content used in each DNA standard by the total numberof cytosines, the % 5-mC of the sample as it relates
to total cytosine content can be determined in therange of 1% to 13% methylation. See the values to theright for the specific % 5-mC for each DNA Standard.

STD 0

0%

STD 101.3%

STD 20

2.6%

STD 303.9%

STD 50

6.6%

STD 75

9.9%

STD 100

13.1%

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14% 5-mC4.

After selecting a method for % 5-mC determination, use a graphing program containing
curve fitting tools to plot the % 5-mC associated with each DNA Standard along the x-axis
and the Net readings along the y-axis. Since the OD values will vary with color development
time, the best fit curve needs to be determined for each experimental run. Extrapolate the %5-mC methylation for each DNA sample. Readerfit (https://readerfit.com) is a freely available
curve fitting program that may be used for analysis.
Note:

If the amount of sample DNA used within the assay was a value other than thesuggested 100 ng DNA/well, the extrapolated value of the % 5-mC should be
divided by the mass of the input sample to achieve a corrected % 5-mC for that
DNA sample. For example, if the 100 ng Standard Curve was used to extrapolate
the % 5-mC for a DNA sample that only contained 50 ng/well, then the extrapolated % 5-mC values are divided by 50 and multiplied by 100% to predict therelative level of methylation in the 50 ng sample.

Figure 2: Graphical representation of the best curve fit as determined by analysis software.
The % 5 methylcytosine represented by each DNA standard was plotted on the x-axis and the Net OD 450nm readings forboth the standards (triangles) and samples (circles) were plotted on the y-axis. The best curve fit was determined and the %5-mC values for the DNA samples were extrapolated from the curve.

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15

Appendix
Section I: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal in
all wells

Omission of key
reagent

Check that all reagents have been added in all wells in thecorrect order

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing a smallaliquot of HRP and Developing Solution together

Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction. Follow ourrecommendations to prepare buffers

Plate reader settings
not optimal

Verify the wavelength and filter settings in the plate reader

Incorrect assay
temperature

Bring Developing Solution and Stop Solution to room temperature before using

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Developing time too
long

Stop enzymatic reaction as soon as the positive wells turn
medium-dark blue or the blank wells turn a faint blue color

Concentration of antibodies is too highIncrease antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations. Make sure to perform the 10 minute incubations in Buffer W during Section D, step 4. Failure to incubate
the washes may increase the background signal.

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations. Ensure no residual wash buffer remains priorto addition of developing solution

Well cross-contamination

Follow washing recommendations

Inaccurate pipetting

Verify that the same amount of hybridized sample, primary
antibody, secondary antibody, developing and stop solutions areadded to each well

Too much sample
per wellDecrease amount of sample per well. Titrate the amount ofgenomic DNA to use per well such that it falls within the rangeof the DNA standards. We suggest a range of 10-200 ng/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working concentration. Start using 1:50 for primary antibody and 1:10 for the secondary antibody. The sensitivity of the assay will be decreased

No signal or weak signal
in sample wells

Not enough sample
per wellIncrease amount of sample per well. Titrate the amount ofgenomic DNA to use per well such that it falls within the rangeof the DNA standards. We suggest a range of 10-200 ng/well

No signal or weak signal
in standard curve wells

Omission of key
reagent

Ensure that all reagents have been added to the wells in thecorrect order. Verify that DNA standards were prepared as
instructed in Step B of the manual.

High background in all
wells

Uneven color
development between
replicates

Signal in sample well beyond the detectable range
for the microplate reader

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16Section J. Sample-specific DNA standards
The provided Methylated and Non-methylated DNA standards included in the assay can be used
to generate a standard curve for relative quantitation. These DNA standards are prepared fromhuman Jurkat genomic DNA to provide researchers known quantities of LINE-1 methylation thatcan be used to approximate the level of 5-mC methylation in their DNA samples. However, thecopy number of the LINE-1 repeat element may vary between sample types. To improve the accuracy of the methylation quantitation based on copy number variances, customers could prepare astandard curve mixture using genomic DNA from their specific cell line or tissue of interest.
i.

Isolate genomic DNA of interest. Reserve 10 ng of DNA for use in preparing the nonmethylated DNA standard and 1 µg to perform the assay. Use 4 µg of the remaining
DNA to prepare the methylated DNA standard.

ii.

Fully methylated one fraction of the genomic DNA using M.SssI enzyme following the
manufacturer’s instructions.

Note:

We have found the following conditions improve the methylation reaction:
Use 2 units M.SssI enzyme (4 U/µl) per µg DNA and 640 µM final concentration of the methyl donor SAM. Incubate the reaction at 37°C for 2 hours andthen spike in additional SAM (the same amount used initially) and continue toincubate at 37°C for another 2 hours.

iii.

Fragment the fully methylated genomic DNA using MseI enzyme.

iv.

Quantify the fragmented fully methylated genomic DNA. Dilute to 10 ng/µl in sterilewater. This DNA can now be used as the Methylated DNA standard to generate thesample-specific standard curve.

v.

Prepare non-methylated DNA standards by performing whole-genome amplification of
the 10 ng fraction reserved of the isolated genomic DNA. Amplification will remove any
endogenous methylation to generate a non-methylated pool of DNA. Since the DNA
is generated by PCR amplification it will already be present in fragments. The DNA
prepared by whole genome amplification does not require additional MseI digestion.

vi.

Quantify the whole-genome amplified genomic DNA. Dilute to 10 ng/µl in sterile water.
This DNA can now be used as the Non-Methylated DNA standard to generate thesample-specific standard curve.

Follow the recommendations for standard curve preparation in the manual using the samplespecific standards. Analysis of DNA samples from the same cell type as the standard curve will
normalize for any copy number variances during quantitation of 5-mC levels.

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17Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

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18