Global 5-hmC Quantification(version A1)

Catalog No. 55018

Active Motif North America
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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.
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The manufacturer of this documentation is Active Motif, Inc.
© 2015 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
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TABLE OF CONTENTS

Page

Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 2Introduction  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Traditional Methods to Study Hydroxymethylcytosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Performance and Benefits  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6Global 5-hmC Quantification Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Kit Components and Storage .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 7Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section A. MseI Digestion of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section B. Plate Set Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section C. Plate Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section D. Standard Curve Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Section E. DNA Sample Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section F. . Binding to the Coated Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section G. Binding of Primary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section H. Binding of Secondary Antibody  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section I. . Colorimetric Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section J. . Analysis of Results  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Appendix
Section K. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
Section L. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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Overview
The Global 5-hmC Quantification Kit is designed to detect and quantify 5-hydroxymethylcytosine(5-hmC) in DNA fragments. 5-Hydroxymethylcytosine is an oxidative product of 5-methylcytosine(5-mC) that is catalyzed by the ten eleven translocation (TET) family of enzymes. Research has
shown that 5-hmC levels are independent of 5-mC levels and both DNA modifications appear
to have distinct biological functions. 5-hmC is known to play a role in transcriptional regulationand embryonic development. It may also serve as a prognostic indicator in certain cancers andneurodegenerative disorders.
In the Global 5-hmC Quantification Kit, genomic DNA of interest is fragmented by enzymatic
digestion and heat denatured to create ssDNA. 96-stripwell plates are coated with a DNA binding
agent to enhance the capture of DNA fragments to the plate. Following the addition of ssDNA
to the coated wells, unbound DNA fragments are washed away. A primary antibody specific for
5-hmC and a secondary antibody conjugated to horseradish peroxidase (HRP) are used for detection of hydroxymethylated fragments. The colorimetric readout is easily quantified by spectrophotometry using a microplate reader at 450nm.
Each kit contains an optimized protocol and reagents necessary to perform DNA fragmentation,plate coating, capture, and colorimetric detection of 5-hydroxymethylcytosine. For added convenience, DNA standards containing known levels of 5-hmC are included in the kit. By generating astandard curve, the 5-hmC levels in each DNA sample can be determined.

product

formatcatalog no.

Global 5-hmC Quantification Kit

1 x 96 rxns

55018

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1Flow Chart of Process
21

11

01

9

8

7

6

5

4

Coat 96-well assay
plate with DNA
Binding Agent

3

2

1
A
B

C
D
E
F
G
H

1 hour at RT(or overnight at 4˚C)

Heat denature
DNA Standards and
MseI digested genomic
DNA samples
10 min. at 98˚C
12

11

10

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

21

11

01

9

Add DNA Standards
and Samples tothe coated plate
2 hours at 37˚C
8

7

6

5

4

3

2

1
A
B

C

Wash to remove
unbound DNA

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-hmC
antibody
1 hour at RT

H

21

11

01

9

8

7

6

5

4

3

2

1
A

Add anti-IgG
HRP conjugate

B
C
D
E
F
G
H

1 hour at RT
5

4

3

2

1

7

6

8

9

10

11

12

A
B
C
D
E
F
G
H

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2

Add developing
and stop solution< 5 min.

Introduction
DNA Modification 5-Hydroxymethylcytosine (5-hmC)
In mammals and other vertebrates, DNA methylation usually occurs at the carbon 5 (C5) positionof cytosine (5-mC), mostly within CpG dinucleotides. In 2009, Kriaucionis and Heintz and Tahiliani
et al. discovered another DNA modification, 5-hydroxymethylcytosine (5-hmC), which wasobserved to be elevated in neurons and embryonic stem cells1,2. The 5-hydroxymethylcytosine
modification results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of cytosine oxygenases2,3. While the precise function of 5-hmC has yet
to be determined, it has been postulated that it could represent a pathway to demethylate DNA,
as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine3. Alternatively, 5-hmC may be produced by the addition of formaldehyde to DNA cytosines
by DNMT proteins4.
DNA base modifications are dynamic marks and their relative concentration at given loci is infludenced by multiple factors including cell and tissue type, the differentiation state of a cell, andthe balance between cellular homeostasis and stress response. 5-hmC has been shown to havedistinct biological functions and cellular localizations from 5-mC. Proteins that bind specifically to
5-hmC include DNA repair factors, splicing mediators and transcription regulators5,6. Experimental
evidence indicates that 5-hmC plays a role in transcriptional regulation and embryonic development7-17.
Loss of global hydroxymethylation is associated with malignant human cancers such as melanoma.
Moreover, mutations in TET2 are commonly observed in a wide range of hematopoietic malignancies and are associated with poor patient outcomes18-20. Hydroxymethylation is also most abundant in the brain, where it is believed to play an important role in neurological development andmemory formation10, 21-23. Therefore, 5-hmC may serve as a prognostic indicator in certain cancers
and neurodegenerative disorders, such as Alzheimer’s disease, Freidrich’s ataxia and Huntington’sdisease.
Using Active Motif’s Global 5-hmC Quantification Kit, genomic DNA from different sample types(e.g. normal and diseased), treatment conditions, clinical outcomes, or environmental backgrounds
can be analyzed for differences in global 5-hydroxymethylcytosine levels. DNA standards areincluded in the kit to prepare a standard curve for sample quantification. The assay can detect as
little as 0.02% 5-hmC. Results obtained with the Global 5-hmC Quantification Kit enable higher
throughput processing of samples, require only 10-50 ng of genomic DNA per well and results are
consistent with mass spectrometry values for the same DNA samples.

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3

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.

Kriaucionis, S. and Heintz, N. (2009) Science, 324: 924-929.
Tahiliani, M. et al. (2009) Science, 324: 930-935.
Huang, Y. et al. (2010) PLoS One, 26: e8888.
Liutkeviciute, Z. et al. (2009) Nat. Chem. Biol., 5: 400-402.
Iurlaro, M. et al. (2013) Genome Biology, 14(10): R119.
Spruijt, C. et al. (2013) Cell, 152(5): 1146-1159.
Gan, H. et al. (2013) Nature Communications, 4: 1995.
Tsumagari, K. et al. (2013) Epigenetics, 8(3): 317-332.
Nestor, C. et al. (2012) Genome Research, 22: 467-477.
Rudenko, A. et al. (2013) Neuron, 79(6): 1109-1122.
Blaschke, K. et al. (2013) Nature, 500(7461): 222-226.
Klug, M. et al. (2013) Genome Biology, 14(5): R46.
Bocker, M. et al. (2012) Nature Communications, 3: 818.
Ficz, G. et al. (2013) Cell Stem Cell, 13(3): 351-359.
Gao, Y. et al. (2013) Cell Stem Cell, 12(4): 453-469.
Piccolo, F. M. et al. (2013) Molecular Cell, 49(6): 1023-1033.
Hackett, J. A. et al. (2013) Science, 339(6118): 448-452.
Lian, C. G. et al. (2012) Cell, 150(6): 1135-1146.
Song, S. J. et al. (2013) Cell, 154(2): 311-324.
Huang, H. et al. (2013) Proc Natl Acad Sci USA, 110(29): 11994-11999.
Colquitt, B. M. et al. (2013) Proc Natl Acad Sci USA, 110(36): 14682-14687.
Lister, R. et al. (2013) Science, 341(6146): doi: 10.1126/science.1237905.
Kaas, G. A. et al. (2013) Neuron, 79(6): 1086-1093.

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4

Traditional Methods to Study Hydroxymethylcytosine
To date, there are several methods used for Hydroxymethylcytosine analysis:
1. Mass Spectrometry (LC-MS/MS): DNA is digested into single nucleosides which are separated according to size. Cytosines, methylated cytosines and hydroxymethylated cytosines
are quantified. Variants of this approach include high performance liquid chromatography(HPLC), as well as two dimensional thin layer chromatography and high performance capillaryelectrophoresis. While these methods are highly quantitative, they require large amounts of
DNA and are not easily amenable to high throughput.
2. Oxidative Bisulfite Sequencing (OxBS-seq): Bisulfite conversion consists of the treatmentof genomic DNA with sodium bisulfite, leading to deamination of unmethylated cytosines
into uracil. With OxBS-Seq, an additional oxidative step is used to discriminate between
5-mC and 5-hmC. The oxidation step converts 5-hmC to 5-formylcytosine (5-fC) which issensitive to deamination by bisulfite. Converted DNA is then PCR amplified and sequenced.
Cytosines that remain in the sequence were 5-mC and not 5-hmC. Ox-BS-Seq enables thediscrimination of 5-mC and 5-hmC with bisulfite conversion, but is used as a readout for
5-mC. It often requires repeated bisulfite treatments to fully deaminate 5-fC and the oxidation can degrade DNA.
3. Tet Assisted Bisulfite Sequencing (TAB-Seq): TAB-Seq utilizes a glucosylation enzyme toprotect 5-hmC in genomic DNA. The DNA is then treated with Tet enzyme to convert 5-mC
to 5-carboxylcytosine (5-caC). The 5-hmC remains protected from oxidation and will be readas cytosines in the sequencing reaction. While TAB-Seq is ideal for specific quantification of
5-hmC, it relies on the activity of the Tet enzyme which tends to be very inefficient.
4. Hydroxymethyl Collector-Seq (hMe-Seal): The Hydroxymethyl Collector-Seq Kit (Active
Motif, Cat 55019) utilizes a glucosylation enzyme to add an azide-modified glucose to 5-hmC.
A biotin is then attached to the modified glucose and streptavidin beads are used to enrichfor DNA fragments containing 5-hmC. This method is highly efficient and specific, but lacks
quantification of 5-hmC.
5. Hydroxymethylated DNA Immunoprecipitation (hMeDIP): Methylated DNA Immunoprecipitation is an immunocapture technique in which an antibody specific for hydroxymethylcytosine is used to immunoprecipitate genomic DNA fragments containing 5-hmC.
Enriched DNA can be used for analysis of the methylation status of a particular gene by PCR,or in combination with NGS. hMeDIP is prone to antibody bias and the technique does notconsider total cytosine content.

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5Kit Performance and Benefits
The Global 5-hmC Quantification Kit is used to quantify 5-hydroxymethylcytosine levels in genomic DNA when comparing the samples to the provided DNA standards.
Range of detection: The dynamic range of the standard curve is 0% to 0.71% 5-hmC. The assay
has been shown to detect as little as 0.02% 5-hmC.
Sample DNA can be used in the range of 10 ng - 50 ng per well. If the OD 450nm readings of thesamples fall outside the limits of detection for the assay (as determined by the standard curve),
we suggest performing a titration of the sample 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 10 - 25 ng/well for DNA samples containing an elevatedconcentration of 5-hmC (e.g. human brain). We recommend a concentration of 25-50 ng/well for
DNA samples containing lower levels of 5-hmC.
Assay time: 5 hours

Global 5-hmC DNA Quantification Kit

0.9
0.8
0.7

% 5-hmC

0.6
0.5
Global 5-hmC ELISA
Mass Spectrometry

0.4
0.3
0.2
0.1
0
mouse mouse
cerebellum heartmouse
kidney

mouse
liver

mouse
lung

human
brain

human humankidney liver

Figure 1: Comparison of 5-hmC quantification using the Global 5-hmC Quantification Kit and mass spectrometry.
The Global 5-hmC Quantification Kit was used to determine the % 5-hydroxymethylcytosine in genomic DNA isolated frommouse and human tissues. Genomic DNA was tested in the range of 20 ng/well (human brain) to up to 50 ng/well (all otherDNA samples). The % 5-hmC was calculated for each sample using the included DNA Standards. The data was compared tomass spectrometry data obtained from 500 ng of the same DNA samples. Results show the Global 5-hmC Quantification
ELISA provides equivalent quantification, while requiring only a fraction of the starting material.

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6

Global 5-hmC Quantification Kit Components and Storage
Global 5-hmC Quantification Kits are for research use only. Not for use in diagnostic procedures.
All components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

MseI enzyme (10 U/µl)

20 µl

-20°C

10X MseI Reaction Buffer

65 µl

-20°C

5-hmC DNA Standards A, B, C, D, E, F & G(10 ng/µl)

25 µl each(set of 7)-20°C

DNA Binding Agent (lyophilized)

2 x 60 mg-20°C

5-Hydroxymethylcytosine pAb

10 µl

-20°C

Anti-rabbit HRP-conj. secondary Ab

10 µl

4°C

Antibody Dilution Buffer

12 ml

4°C

20X Wash Buffer

25 ml

4°C

Developing Solution

10 ml

4°C

96-well plate

1 ea

4°C

Stop Solution

10 ml

RT

10X Sample Dilution 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• Nanodrop, Qubit or equivalent method to determine DNA concentration• Incubator set to 37°C
• Thermal cycler or water bath set to 65°C and 98°C
• Ice-water bath
• Orbital shaker (e.g. Multi-microplate Genie from Scientific Industries)• 1.5 or 2 ml microcentrifuge tubes and 15 ml conical tube
• Microcentrifuge with PCR tube and microcentrifuge tube adapters• Multichannel pipette, 10-200 µl pipette tips and plastic reservoirs• Vortex• DNase-free sterile water• Spectrophotometric microplate reader

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7

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 up to
32 DNA digestions as described in Step A of the protocol. Digested DNA is assayed in duplicate.
10X MseI Reaction Buffer
The reaction buffer is provided ready to use at a 10X concentration. Please follow the instructionsin the protocol for use.
DNA Samples
Following MseI digestion to fragment the DNA samples (as described in Section A), DNA must be
quantified for accurate analysis in the assay. It is recommended to use a range of 10-50 ng/wellof MseI digested DNA in a final volume of 50 µl per well. If you anticipate your sample to containhigher concentrations of 5-hydroxymethylcytosine, such as brain samples, the lower end of therange can be used. For other sample types, we recommend starting with 50 ng/well. It has beenreported that cultured cell lines contain very low levels of 5-hydroxymethylcytosine, therefore,
samples from cultured cell lines may not be suitable for use in analysis of 5-hmC levels.
We recommend preparing samples for testing in duplicate. DNA samples should be diluted in
Sample Dilution Buffer. It is important that your sample fall within the linear range of the DNA
Standards for accurate quantification. It may be necessary to test several DNA concentrations todetermine the optimal amount for your sample type. The following table provides recommended
volumes for preparing samples in duplicate and includes a slight excess to account for pipetting
losses. DNA should be prepared in 0.2ml PCR strip tubes for heat denaturation in a thermal cycler.
Components

10 ng/well

20 ng/well

25 ng/well

50 ng/well

Blank

MseI Digested DNA
10X Sample Dilution Buffer
Distilled water

22 ng
11 µl
Up to 110 µl

44 ng
11 µl
Up to 110 µl

55 ng
11 µl
Up to 110 µl

110 ng
11 µl
Up to 110 µl

–
11 µl
99 µl

A correction factor will be applied during data analysis to compensate for any concentration differences between the samples and the DNA standards.
DNA Binding AgentThe DNA binding agent is provided lyophilized and each vial is sufficient to coat 48-wells of theassay plate. Before opening, quick spin the vial for 1 minute at room temperature to collect contents to the bottom of the tube. Add 1 ml sterile water and vortex until fully dissolved. Transfer
the entire contents of the vial to a 15 ml conical tube. Add 5 ml sterile water to the conical tube
for a total volume of 6 ml and vortex to mix. Use 100 µl/well to coat the assay plate. If coatedplates are not to be used immediately, dried plates can be stored in a foil pouch with a desiccant
at 4°C and used within 2-weeks. Discard any unused DNA binding agent solution.
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8

5-hmC DNA Standards
DNA Standards are provided a concentration of 10 ng/µl. Standards are provided as dsDNA andmust be heat denatured before use in the assay. Each DNA standard contains DNA with a known
quantity of 5-hydroxymethylation. For quantification of 5-hmC levels in the samples, the entire
range of DNA standards should be used to create a standard curve. Sample results are thencompared to the DNA standard curve to extrapolate the % 5-hmC in each sample. Enough DNA isprovided to perform two standard curves in duplicate.
To improve accuracy of quantification, we recommend using the DNA standards at a concentrationequivalent to the amount of test sample. If working with samples containing a high abundance of
5-hmC (e.g. brain), we suggest using DNA standards at a concentration of 20 ng/well. If working
with samples containing lower amounts of 5-hmC, we suggest using DNA standards at a concentration of 50 ng/well. A correction factor will be applied during data analysis to compensate forany concentration differences between the samples and the DNA standards.
1X Buffer W (See the Quick Chart for Preparing Buffers)
Prepare the amount of 1X Buffer W required for the assay. For every 10 ml of 1X Buffer W required,dilute 1 ml 10X Buffer W with 9 ml sterile water.
Antibody Dilution Buffer
The antibody dilution buffer is provided ready to use. Pre-warm to room temperature before use.
5-Hydroxymethylcytosine pAb
Prepare a 1:2,000 dilution of the 5-hydroxymethylcytosine polyclonal antibody into Antibody Dilution Buffer by performing a two-step dilution. First, prepare a 1:10 dilution into Antibody Dilution
Buffer. Then prepare a 1:200 dilution into Antibody Dilution Buffer for the required amount ofantibody needed for the experiment. Use 50 µl per well.
Components

1 strip(8 wells)

4 strips(32 wells)

6 strips(48 wells)

12 strips(96 wells)

Initial 1:10 Dilution
5-Hydroxymethylcytosine pAb
Antibody Dilution Buffer
Total Volume

2 µl
18 µl
20 µl

2 µl
18 µl
20 µl

2 µl
18 µl
20 µl

4 µl
36 µl
40 µl

Secondary 1:200 Dilution
5-hmC pAb pre-diluted 1:10
Antibody Dilution Buffer
Total Required

2.25 µl
448 µl450 µl

8.5 µl
1.692 ml
1.7 ml13 µl2.587 ml
2.6 ml26 µl5.174 ml
5.2 ml

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 10 ml of 1X
Wash Buffer required, dilute 0.5 ml 20X Wash Buffer with 9.5 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. Use the 1X Wash Buffer at room temperature.
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9Anti-rabbit HRP-conj. secondary antibody (See the Quick Chart for Preparing Buffers)
Prepare a 1:4,000 dilution of anti-rabbit HRP-conj. secondary antibody into Antibody Dilution Buffer. We recommend performing a two-step dilution by first preparing a 1:10 dilution into Antibody
Dilution Buffer. Then prepare a 1:400 dilution into Antibody Dilution Buffer for the required
amount of antibody needed for the experiment. Use 50 µl per well.
4 strips(32 wells)

6 strips(48 wells)

12 strips(96 wells)

Initial 1:10 Dilution
Anti-rabbit HRP-conjugated antibody 2 µl
Antibody Dilution Buffer
18 µl
Total Volume
20 µl

2 µl
18 µl
20 µl

2 µl
18 µl
20 µl

2 µl
18 µl
20 µl

Secondary 1:400 Dilution
Anti-rabbit HRP pre-diluted 1:10
Antibody Dilution Buffer
Total Required

4.25 µl
1.696 ml
1.7 ml

6.5 µl
2.594 ml
2.6 ml13 µl5.187 ml
5.2 mlComponents

1 strip(8 wells)

1.125 µl
449 µl450 µlDeveloping 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 lightduring storage. The Developing Solution may develop a yellow hue over time. This does not affectproduct 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 forthe assay 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 any remaining Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.

www.activemotif.com

10Quick Chart for Preparing Buffers
Reagents to prepare Components

For 1
well

For 1 strip For 4 strips(8 wells) (32 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

1X Buffer W

Distilled water
10X Buffer W
TOTAL REQUIRED

900 µl100 µl1 ml

8.1 ml900 µl9 ml

31.5 ml
3.5 ml
35 ml

45 ml
5 ml
50 ml

90 ml
10 ml
100 ml

1X Wash Buffer

Distilled water
20X Wash Buffer
TOTAL REQUIRED

950 µl50 µl
1 ml

8.55 ml
450 µl9 ml

33.25 ml
1.75 ml
35 ml

47.5 ml
2.5 ml
50 ml

95 ml
5 ml
100 mlDeveloping Solution

TOTAL REQUIRED

100 µl900 µl3.5 ml5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

100 µl900 µl3.5 ml5.4 ml10.8 mlwww.activemotif.com

11

Assay Protocol
Read the entire protocol before use.
Prior to starting the Global 5-hmC Quantification Kit, genomic DNA is fragmented using restriction
digestion with the provided MseI enzyme. This enzymatic digestion will create the appropriate
size range of DNA fragments for optimal detection in the assay.

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 Global 5-hmC Quantification assay can be performed on
approximately 10-50 ng MseI fragmented genomic DNA per well. We suggest using 0.5 µg genomic
DNA per sample type for digestion and then performing the assay in duplicate.
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 (0.5 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 2-4 hours.

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. The
Global 5-hmC Quantification assay is very sensitive to DNA concentration so DNA MUST bequantified 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.

www.activemotif.com

12Section B: Plate Set-Up
Determine the appropriate number of microwell strips required for testing samples, standards andblank wells in duplicate. Remove the unused strips from the strip holder and place in the aluminum pouch at 4°C. Allow all microwell strips to be used in the assay to warm to room temperature.
If less than 8 wells in a strip need to be used, cover the unused wells with a portion of the adhesive plate sealer while you perform the assay. The unused wells are stable at room temperature forthe duration of the assay if kept dry. Once the assay is finished, unused wells should be returnedto the aluminum pouch and stored at 4°C.
To improve the binding efficiency of the genomic DNA to the assay plate, a DNA Binding Agent isapplied to each well. The DNA Binding Agent is provided lyophilized and each vial is sufficient tocoat 48-wells of the assay plate. If not used immediately, coated plates should be dried and stored
in a foil pouch with a desiccant at 4°C and used within 2-weeks.
Please plan your experimental design and plate coating accordingly. If only a portion of the plate
will be coated and used in the assay, be sure to designate whether the stored strips are coated ornot. An example plate layout is shown below.
DNA Standards

MseI-digested Samples

1

2

3

4

5

6

A

STD A

STD A

Sample 1

Sample 1

Sample 9

Sample 9

B

STD B

STD B

Sample 2

Sample 2

Sample 10 Sample 10

C

STD C

STD C

Sample 3

Sample 3

Sample 11

D

STD D

STD D

Sample 4

Sample 4

Sample 12 Sample 12

E

STD E

STD E

Sample 5

Sample 5

Sample 13 Sample 13

F

STD F

STD F

Sample 6

Sample 6

Sample 14 Sample 14

G

STD G

STD G

Sample 7

Sample 7

Sample 15 Sample 15

H

Blank

Blank

Sample 8

Sample 8

Sample 16 Sample 16

Sample 11

Section C: Plate Coating
Coat the appropriate number of wells as determined in the plate set-up. Each DNA Binding Agentvial is sufficient to coat 48 wells. Coated plates can stored at 4°C and used within 2-weeks.
1.

Allow microwell strips to warm to room temperature.

2.

Reconstitute the DNA Binding Agent in water and prepare 1X Buffer W as described in thesection Buffer Preparation and Recommendations.

3.

Add 100 µl of the reconstituted DNA Binding Agent to each well. Cover the wells with theadhesive plate sealer. Incubate at room temperature for 1 hour. (Or overnight at 4°C.)
www.activemotif.com

13

4.

Remove the plate sealer and discard the DNA Binding Agent from the wells of the plate byinverting the plate over a liquid waste receptacle. Then, tap the inverted plate 3 times on
absorbent paper towels.

5.

Add 150 µl 1X Buffer W to each well and immediately discard by inverting the plate over aliquid waste receptacle. Then, tap the inverted plate 3 times on absorbent paper towels.

6.

Repeat the wash 2 more times for a total of three washes.

7.

Tap the inverted plate 3-5 times on absorbent paper towels to remove all the liquid. Failure
to remove all the buffer at this step will lead to inaccurate 5-hmC quantification. Plate can beallowed to air dry for up to 2 hours. If the strips will not be used immediately, we recommend letting them air dry for 4 hours before storing in the foil pouch with a dessicant.

8.

While the plate is drying, prepare the DNA Standards and Samples as described in Section D& Section E.

Section D: Standard Curve Preparation
The provided 5-hmC DNA Standards can be used to create a standard curve in duplicate for quantification of percent hydroxymethylcytosine in each sample. Use the entire range of standards asshown in the table below. DNA standards should be prepared at a concentration equivalent tothe amount of sample to be tested. For optimal accuracy in quantification, we suggest using DNA
standards at 20 ng/well for samples with a high abundance of 5-hmC and we suggest using DNA
standards at 50 ng/well for samples with a low abundance of 5-hmC. A correction factor will beapplied during data analysis to account for any differences between the quantity of the samples
and the DNA standards.
1.

Prepare a labeled 0.2 ml stripwell PCR tubes for heat denaturation of the DNA standards.
Add the appropriate volume of DNA standard and sterile water to each tube to achieve thedesired standard curve concentration as described in the chart below.
20 ng/well

50 ng/well% 5-hmC

Volume of
DNA Standard

Volume of
Water

Volume of
DNA Standard

Volume of
Water

STD A

0.71%

4.4 µl105.6 µl

11 µl

99 µl

STD B

0.36%

4.4 µl105.6 µl

11 µl

99 µl

STD C

0.18%

4.4 µl105.6 µl

11 µl

99 µl

STD D

0.09%

4.4 µl105.6 µl

11 µl

99 µl

STD E

0.045%

4.4 µl105.6 µl

11 µl

99 µl

STD F

0.022%

4.4 µl105.6 µl

11 µl

99 µl

STD G

0%

4.4 µl105.6 µl

11 µl

99 µl

www.activemotif.com

14

2.

Vortex to mix thoroughly. Quick spin to collect liquid.

3.

Heat denature samples in a thermal cycler programmed to heat the samples from 25°C to
98°C for 10 minutes. Immediately plunge samples into an ice-water bath for 5 minutes. (Denaturation can be performed in combination with DNA samples prepared in Section E.)

Section E: DNA Sample Preparation
It is recommended to use a range of 10-50 ng/well of MseI digested DNA in a volume of 50 µl per
well. If you anticipate your sample to contain higher concentrations of 5-hydroxymethylcytosine, such as brain samples, the lower end of the range can be used. For other sample types, werecommend starting with 50 ng/well. A correction factor will be applied during data analysis tocompensate for any concentration differences between the samples and the DNA standards.
1.

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

2.

Add DNA to the appropriately labeled PCR tube. (e.g. For duplicate reactions, add 110 ng
digested DNA diluted in Sample Buffer in a final volume of 110 µl.)3.

Prepare blank wells as described in the DNA Sample Preparation Table on page 8.

4.

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

5.

Place samples in a thermal cycler and incubate at 98°C for 10 minutes. Immediately plunge
samples into an ice-water bath for 5 minutes.

Section F: Binding to the Coated Plate
1.

Quick spin the heat denatured DNA standards and samples to collect any condensate.

2.

Add 50 µl of each DNA standard, sample and blank to the coated assay wells according to
the plate layout. Ensure the bottom of the wells are completely covered.

3.

Cover the plate with adhesive plate sealer and incubate at 37°C for 2 hours.

4.

During this incubation, prepare the 5-Hydroxymethylcytosine antibody dilution as describedin the section Buffer Preparation and Recommendations.

5.

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

6.

Add 150 µl Buffer W to each well and immediately discard as described above.

7.

Repeat the wash 2 more times for a total of three washes.

Section G: Binding of Primary Antibody
1.

Add 50 µl of diluted 5-Hydroxymethylcytosine antibody to each well being used, including
blank wells. Cover the plate with plate sealer and incubate for 1 hour at room temperature
with mild agitation (200-300 rpm on an orbital shaker).
www.activemotif.com

15

2.

During this incubation, prepare the 1X Wash Buffer and the anti-rabbit HRP antibody dilution
as described in the section Buffer Preparation and Recommendations.

3.

After the incubation, rinse the wells 3 times with 150 µl 1X Wash Buffer.

Section H: Binding of Secondary Antibody
1.

Add 50 µl of diluted HRP-conjugated anti-rabbit antibody to each well being used, including
blank wells. Cover the plate with plate sealer and incubate for 1 hour at room temperature
with mild agitation (200-300 rpm on an orbital shaker).

2.

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

3.

After the incubation, rinse the wells 3 times with 150 µl of 1X Wash Buffer.

Section I: 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 from 1 to 3 minutes at room temperature protected from direct light. Please readthe Certificate of Analysis supplied with this kit for optimal development time associatedwith this lot number. Monitor the blue color development in the standard curve wells containing the highest concentrations of hydroxymethylated DNA (STD A) until they turn lightto medium blue. The Blank wells should remain clear. 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 yourplate reader does not have this capability, you may read the plate twice, once at 450 nm andonce at 655 nm then manually subtract the 655 nm OD from the 450 nm OD values.

Section J: Analysis of Results
1.

Average the duplicate 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.

The Blank wells represent the baseline signal from the assay reagents in the absence of DNA.
The STD G (0% 5-hmC) value displays the background signal of the assay in the presence ofunmethylated DNA samples, while the STD A represents a sample containing 0.71% 5-hmC.
www.activemotif.com

16

3.

For quantitative analysis, plot the % 5-hmC of the DNA Standards along the X-axis and the
Net readings for the DNA Standards along the Y-axis. Draw the best curve fit using a graphing
software program (e.g. Excel Linear Regression), using only the standards in the linear portionof the curve. Since the OD values will vary with color development time, the best curve fit
needs to be determined for each experimental run. Extrapolate the % 5-hmC for each DNA
sample from the curve.

4.

Apply a correction factor to adjust the values for any samples that were run at a different
concentration than the DNA Standards.

			(DNA Standard concentration in ng/well)
Adjusted % 5-hmC = (Calculated % 5-hmC) x
			(Sample Quantity in ng/well)
Example calculation:
Sample Quantity: 10 ng/well human brain genomic DNA
Net OD 450nm:
0.645Standard curve:
See Figure 2 below y=1.5131(x) + 0.0277
Calculated % 5-hmC:			
0.645 = 1.5131(x) + 0.0277
					
x = 0.408 or 0.408%
				

Adjusted % 5-hmC = (0.408%) x

20 ng/well
10 ng/well= 0.82% 5-hmC

Figure 2: Graphical representation of the best curve fit as determined by analysis software.
The % 5 hydroxymethylcytosine represented by each DNA standard was plotted on the x-axis and the Net OD 450nm readings for the DNA standards were plotted on the y-axis. The best curve fit was determined using Excel Linear Regression.
The calculated % 5-hmC values for the DNA samples were extrapolated from the curve using the trendline equation.

www.activemotif.com

17

Appendix
Section K: 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.

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 sample, primary antibody,
secondary antibody, developing and stop solutions are added
to each well

Signal in sample wellbeyond the detectable
range for the microplate
readerToo 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 10-50 ng/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working
concentration. Start using 1:4000 for primary antibody and
1:5000 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 10-50 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 Section D of the manual.

High background in all
wells

Uneven color
development between
replicateswww.activemotif.com

18Section L: Related Products
DNA Methylation		

FormatCatalog No.

MethylDetector™		
MethylCollector™ Ultra		
HypoMethylCollector™		
Hydroxymethyl Collector		
MeDIP		hMeDIP		
DNMT Activity / Inhibition Assay		
Methylated DNA Standard Kit		
5-Carboxylcytosine DNA Standard Kit		
Fully Methylated Jurkat DNA		
Recombinant Tet1 protein		b-Glucosyltransferase enzyme		
PvuRts1I restriction enzyme		

50 rxns
30 rxns
30 rxns
25 rxns
10 rxns
10 rxns
1 x 96 rxns
3 x 2.5 µg
0.5 µg
10 µg
25 µg
500 Units
50 Units

55001
55005
55004
55013
55009
55010
55006
55008
55014
55003
31363
55012
55011

Whole Genome Amplification		

FormatCatalog No.

GenoMatrix™ Whole Genome Amplification Kit		

50 rxns

58001

Antibodies

Application

FormatCatalog No.

DB
DB, IF
DB, IF
DB, IF, IHC, MeDIP
DB, MeDIP
FACS, IHC, IP, MeDIP
DB, MeDIP
ChIP, IHC, IP, WB
WB
ChIP, IF, IHC, WB
ChIP, IF, IP, WB
WB
WB
WB
WB
WB
WB

100 µg100 µl100 µl100 µl100 µg50 µg100 µg100 µg100 µg100 µg100 µg100 µl100 µg100 µg100 µg100 µg100 µg

61111
61225
61223
39769
39999
39649
61255
39204
39205
39206
39207
39907
39215
40965
39216
39217
39218

3-Methylcytosine (3-mC) pAb
5-Carboxylcytosine (5-caC) pAb
5-Formylcytosine (5-fC) pAb
5-Hydroxymethylcytosine pAb
5-Hydroxymethylcytosine mAb
5-Methylcytosine mAb
5-Methylcytosine pAb
DNMT1 mouse mAb
DNMT2 rabbit pAb
DNMT3A mouse mAb
DNMT3B mouse mAb
DNMT3L rabbit pAb
MBD1 mouse mAb
MBD2 mouse mAb
MBD3 mouse mAb
MBD4 mouse mAb
MeCP2 rabbit pAb

Active Motif also offers a growing list of application validated antibodies, including antibodies
for histones and histone modifications, transcription factor antibodies, DNA methylation-related
antibodies and ChIP validated antibodies. For a complete list go to www.activemotif.com/abs
Histone Purification		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		

10 rxns
20 rxns

40025
40026

Chromatin Assembly		

FormatCatalog No.

Chromatin Assembly Kit		
HeLa Core Histones		

10 rxns
36 µg

53500
53501

www.activemotif.com

19Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008, USA
E-mail: tech_service@activemotif.com

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351

Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Active Motif China
787 Kangqiao Road
Building 10, Suite 202
Pudong District
Shanghai, 201315, China

Direct: (86)-21-20926090
Hotline: 400-018-8123
E-mail: techchina@activemotif.com

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20