Microarrays and DNA methylation profiling
Dr.
Axel Schumacher © 2004-2007
The method described in detail
below is based on the analysis of the enriched unmethylated DNA fraction, using
a series of treatments with methylation-sensitive restriction enzymes, adaptor
ligation, PCR amplification, and quantitative mapping of unmethylated DNA
sequences using microarrays. The key advantages of this approach are the
ability to investigate DNA methylation patterns using very small DNA amounts
and also relatively high informativeness in comparison to the other restriction
enzyme- based strategies for DNA methylation profiling (1).
1. Introduction

Fig 1: Schematic outline of the microarray-based method for identification of
DNA methylation differences in genomic DNA. Samples are cleaved by
methylation-sensitive restriction endonucleases, such as HpaII and Hin6I,
ligated to the CpG-overhang specific adapter, and then cut by McrBC to
eliminate residual methylated DNA fragments. The resulting unmethylated DNA
fragments are then selectively enriched by adapter-specific aminoallyl-PCR,
labelled, and hybridized to microarrays.
2. Materials
2.1 Adapter design
- Adapter storage buffer ST: Prepare 100 ml of 10
mM Tris, pH 8.5; 50 mM NaCl.
- Primer for
preparation of universal-adapter CG-1: primer CG-1a:
5’-CGTGGAGACTGACTACCAGAT-3’ and primer CG-1b: 5’-AGTTACATCTGGTAGTCAGTCTCCA-3’.
2.2
Methylation-sensitive cleavage of DNA
1.
Restriction
enzymes: 10 U/µl HpaII (Fermentas, Lithuania) and 10 U/µl Hin6I (Fermentas). (Note: HhaI is an isoschizomer of Hin6I,
however, the HhaI restriction enzyme is not suitable
for adapter-based methylation analyses, since this enzyme does not produce a
CpG-overhang that can be ligated to the adapter).
2.
Spike DNA (optional; see Methods)
2.3 Ligation
- Prepare a 10 µM ATP stock-solution. Make aliquots
and store at -20°C.
- T4 DNA Ligase (Fermentas, Lithuania)
2.4 McrBC digestion
- McrBC enzyme from New England BioLabs
(NEB).
- Guanosine triphosphate (GTP; is
delivered with the enzyme): make 10x aliquots, since GTP is highly
unstable.
2.5 CpG-specific adapter amplification
- Labelling nucleotides [20 mM]: Thaw one vial aminoallyl (aa)-dUTP
from Ambion, Austin, TX (50 µl solution 5-(3-aminoallyl)-2'-deoxyuridine
5'-triphosphate). Add 16.5 µl H2O
and from 100 mM nucleotide stock solutions (Fermentas) add 16.6 µl dTTP, 41.6 µl dGTP, 41.6 µl dATP, and 41.6 µl dCTP. Mix
and store at -20°C.
- Taq polymerase [5 U/µl]
(NEB).
- 200 pmol/µl CG-1b-primer (see 2.1).
2.6 Purification of amplification product
- Microcon YM-50 columns from Millipore.
2.7 Labelling
- Sodium bicarbonate buffer (0.1 M, pH 9.0): Prepare 10
ml of a 0.1 M Na2CO3 solution. Add 0.42 g of NaHCO3
to another flask. Add 50 ml of water, and mix with a magnetic stirrer
until the NaHCO3 dissolves. The pH should be around 8.5. Adjust
the pH to 9.0 by adding ~ 2.5-3.0 ml of the 0.1 M Na2CO3
solution. Monitor the pH change carefully and do not exceed pH 9.3.
Filter-sterilize the buffer,
which should be prepared as fresh as possible. Aliquot and store at –20°C. Do not re-freeze the buffer. Use every aliquot only once.
- Prior to
coupling, prepare the dyes (FluoroLink
monofunctional dye, Cy3 and Cy5, Amersham Biosciences). The Cy-dyes come
in packages, which contain 5 vials. Dissolve 1 vial of dye in 72 µl DMSO.
Aliquot 4.8 µl in 15 light protected amber reaction tubes, dry immediately
in a speedvac and store at –20°C protected from
light. (H2O is also
used to dissolve Cy-dyes, however the mono
reactive ester is labile in water. Do not use Dioxane, which was suggested
elsewhere as an alternative to DMSO. It appears that Cy5 is not fully
soluble in Dioxane. DMSO on the other hand is hygroscopic and will absorb
moisture from the air, which will react with the NHS ester of the dye and
significantly reduces the coupling reaction efficiency. Therefore, keep
the DMSO supplied in an amber screw-capped vial at -20°C, and let the vial
warm to room temperature before opening to prevent condensation. While the
dyes are usually good when first opened, they are sensitive to moisture
with a short half-life in aqueous environments. Thus take care to store
aliquots of them desiccated, if possible under vacuum).
- Quenching
buffer: Prepare a 10 ml solution of 4 M hydroxylamine (Sigma). Make
aliquots and store at -20°C. Hydroxylamine has top be handled with care,
since it may explode when heated. It is an irritant to the respiratory tract,
skin, eyes, and other mucous membranes. It may be absorbed through the
skin, is harmful if swallowed, and is a possible mutagen.
- Purification of dyes: MiniElute
PCR-purification kit, Qiagen, Hilden/GER.
- Prepare a 3 M NaOAc solution (pH 5.2).
2.9 Hybridization
- Prehybridization buffer:
To 2 ml SlideHyb Glass Array Hybridization Buffer #2 from Ambion add 80 µl of 20 µg/µl yeast tRNA (Sigma) and 200 µg
bovine serum albumin (BSA).
- Hybridization buffer: To
2 ml SlideHyb Buffer #2 from Ambion (other hybridization
buffers may be used. The optimal buffer depends on several experimental
variables such as type of glass slide used, the length and GC content of
the attached nucleic acid targets, and the manufacturing protocol
employed.) Add 50 µl of 20 µg/µl yeast tRNA
(Sigma) and 200 µl
Cot-1 DNA [1 µg/µl] from Roche Diagnostics. The COT fraction of
human placental gDNA consists mainly of repetitive DNA elements. COT DNA
is expensive and hence may be omitted if the microarray used does not
contain larger amounts of repetitive sequences. If using cDNA arrays, 10-20 µg poly(dA)-poly(dT), which blocks
hybridization to polyA tails of cDNA, may be added.
- Wash solution I
(low stringency): prepare 1 litre of 2x SSC, 0.5 % SDS. Filter-sterilize
the buffer. (Note: Wash buffer impurities can cause grainy
background; SDS will fluoresce in the Cy5 channel while salt will
fluoresce in the Cy3 channel. To eliminate this problem use high quality
SDS and SSC for preparation of wash buffers and filter sterilize.
Insufficient washing may also contribute to grainy background, therefore,
increasing the wash times and/or slight agitation may help to decrease the
background).
- Wash solution II
(high stringency): prepare 1 litre 0.5x SSC, 0.5 % SDS. Filter-sterilize
the buffer.
- Wash solution III: 1 litre Isopropanol
(2-propanol). Use only high quality isopropanol (>99.8%). When it is
less concentrated or exposed for a prolonged time to air (it absorbs water
quickly), isopropanol causes streaks or residue on the array surface.
- Cover slips: Hybri-Slips
(Sigma). Hybri-slips have hydrophobic surfaces,
which guarantee a constant probe concentration and, unlike glass cover slips, do not adsorb the probes.
3.
Methods
Here we present a complete
protocol of DNA methylation profiling using the unmethylated fraction of the
genome. This protocol works with most microarray types, as exemplified here
with CpG island- and oligonucleotide microarrays. A detailed description of how
specific ‘epigenetic’ microarrays are designed and how the slides are processed
can be found in Schumacher et al., 2006 (1). DNA samples can either be
interrogated as pairs (tester and control) on one array, as described in this
protocol, or hybridized independently with only one fluorescent dye (e.g. for
Affymetrix tiling arrays).
3.1 Adapter design
In the CpG-specific adapter design, the following aspects must be taken
into account:
- It must
contain a CpG-overhang, which fits to the restriction site of the enzymes
used (Fig. 2a).
- The two
nucleotides next to the CpG-overhang have to be different from the
nucleotides within the recognition sequence of all the enzymes used in the
restriction-digests. This ensures that, after ligation of the adapters,
the old restriction-site is disrupted and the sequence cannot be re-cut by
the enzymes.
- The
melting-temperature (Tm) should be about equal throughout the
adapter and decreasing at the 3’-end opposite to the CpG-overhang.
- The
core-sequence of the adapter should be non-homologous to any sequence in
the gDNA. Additionally, it is advisable to avoid palindromic sequences,
which can lead to mispriming.
- If you want to
cut the adapter from the target-fragments, include a very specific, new
recognition site within the adapters core-sequence (close to
CpG-overhang), which does not cut frequently in the genome you analyze.
Use a rare 8-mer (e.g. SdaI (5’-CC↑TGCA↓GG) for sticky ends or MssI (GTTT↓AAAC) for blunt ends.
- Use a
specific, long antisense-overhang, which
prevents the forming of tandem-repeats and random blunt-end ligation to
the genome (e.g. through degraded DNA).
- To avoid
improper annealing between the two primers, choose a non-5’-complementary
end (Fig. 2a).

Fig. 2: Preparation of the
CpG-specific adapter. a:
CG1-adapter components. b: Annealing of the CG-primers produces
a double-stranded adapter that can be identified by electrophoresis as a strong
band (200 pmol loaded; 2.5 % agarose gel).
3.1.1 Adapter preparation
1.
Dissolve the non-phosphorylated
ssDNA oligonucleotides in ST buffer with a
concentration of approximately 800 pmol. The presence of some salt is necessary
for the oligos to hybridize into a double stranded (ds)-adapter. Keep some of the CG-1b oligo, as it will be
later used as a primer to amplify the unmethylated DNA fraction. Measurethe concentration in a spectrophotometer and adjust
the concentration to 400 pmol. (Note: Quantify in
spectrophotometer. Re member the OD260 = 1 of single stranded DNA is equivalent
to 33 µg/ml not 50 µg/ml! This relationship, however, can be inaccurate for
short fragments of DNA, such as oligonucleotides. Base composition and even
linear sequence will affect optical absorbance, hence
the precise value of the OD to mass relationship is unique for each oligo. For
example, 1.0 OD260 of CCCCCCCCCCCC (homopolymeric deoxycytidine) equals 39 micrograms while 1.0 OD260 of
AAAAAAAAAAAA (homopolymeric deoxyadenosine)
equals only 20 micrograms. Do not believe the amount of primer as indicated by
the manufacqturer. Perform the appropriate
calculation (800 pmol primer = 0.0008 x MW of oligo)).
- Mix together
equal molar amounts of each complementary oligonucleotide to a final concentration
of 200 pmol, and heat 5 min in a thermalcycler at 80°C. Cool down with
1°C/min until the mixture reaches room temperature (RT). The longer the
cooling period, the lower is the risk of hairpin structures.
- Check a small
amount of the ds-Adapter on a 2.5% Agarose-gel
(see Fig. 2b), which should generate a strong band. Run the single
stranded primers next to the adapter as a control.
- Store the
adapters at –20°C.
3.2 Methylation-sensitive
cleavage of DNA
Over 260 different methylation-sensitive restriction
enzymes (MSRE’s; incl. isoschizomers)
are now available, however, not all enzymes are useful
and informative for DNA methylation profiling. Informative MSREs
are defined by the number of cleavage fragments that can be ligated to adapters
and efficiently amplified, and are not lost during column-purification steps (3). Some enzymes, although they cut frequently in the genome, produce
fewer informative fragments compared to enzymes which do not cut as frequently.
For example, the non-palindromic AciI (5’-CCGC-3’) recognizes more than twice as many CpG
sites in CpG island regions when compared to HpaII, but on the other hand, produces fewer fragments in the
size-range that can be detected by PCR or amplified fragment
length polymorphism (AFLP) methods (Table 1).
|
MSRE
|
Cut site 5’-3’
|
~% of CpGs in CpG islands*
|
~% of CpGs in non-CpG islands*
|
Fragments/kb in CpG islands*
|
Fragments/kb in non-CpG islands*
|
|
AciI (SsiI)
|
CCGC,
GCGG
|
30.60
|
17.36
|
3.23
|
1.79
|
|
Hin6IΔ
(HinP1I)
|
GCGC
|
14.40
|
5.05
|
3.98
|
0.61
|
|
HpaII
(BsiSI)
|
CCGG
|
11.70
|
9.33
|
3.98
|
1.18
|
|
Hin1I (BsaHI)
|
GRCGYC
|
2.6
|
0.9
|
1.92
|
0.11
|
|
HpyCH4IV
(TaiI)
|
ACGT
|
1.66
|
6.73
|
1.24
|
0.97
|
|
Bsp119I
(AsuII)
|
TTCGAA
|
0.11
|
0.13
|
0.11
|
<0.02
|
|
Bsu15I
(ClaII)
|
ATCGAT
|
<0.05
|
0.39
|
<0.02
|
0.02
|
Different requirements for the enzyme reduces
the list of potentially useful and informative MSRE’s
to about 17, which would cover up to 85% of all CpG island CpG dinucleotides,
but less than 50% of all CpG dinucleotides in other genomic regions (3). Here, we demonstrate the
technology with a double-digestion using HpaII and Hin6I enzymes (Note: To gain the most out of restriction
analyses, it is crucial to choose the right enzyme combination for the targets
to be interrogated. For example, some MSRE’s cut
relative frequently in CpG islands, but rarely recognize a sequence outside of
a CpG island region, as is the case for Hin6I (5’-GCGC-3’) or Bsp143II
(5’-PuGCGCPy-3’). In contrast, enzymes such as HpyCH4IV (5’-ACGT-3’) cut
predominantly outside of CpG island sequences and are less useful in the
interrogation of CpG islands, for instance in CpG island-microarray based
studies (3). Several other methods rely on the
specific methylation-sensitive cleavage of the rare cutter NotI
(5’-GCGGCCGC-3’), for example RLGS and AFLP methods and a couple of microarray
approaches (6, 7). However, NotI-sites
are not well represented in the genome and will only provide a very rough overview
of methylation patterns. Hence, it is not advisable to include NotI in genome-wide analyses of e.g. complex diseases).
Before the test DNA can be processed, it is
advantageous to add “spiking” DNA to the gDNA samples. The spiking DNA consists
of ‘alien’ (exogenous) DNA, which allows monitoring of each step of the
experiment (Note:
Additionally, spike DNA can be used to test the methylation-sensitive cleavage
reaction, ligation efficiency, McrBC digestion and PCR reaction. Spiking DNA is
added to the reactions with concentrations equivalent to the template
concentration (1 genome equivalent, GE, assuming 3x109 bp
in the human genome). For example, 16.2 pg of λ plasmid is added to 1 µg of template
DNA. Make 12x GE stock solutions in 1 mM EDTA; aliquot into small volumes and
store at -20°C. To prepare a McrBC digestion control, digest pUC57 with HpaII,
ligate the CG-adapter and premethylate with SssI methylase. McrBC controls are
added to the McrBC cleavage step as premethylated plasmid. Other suitable controls
are ФX174
to test for PCR bias or pBR322 to test the ligation efficiency (1). ФX174 DNA has to be digested separately
with HpaII/Hin6I and then ligated to the CG-adapter. Complementary spiking
oligonucleotides have to be representative sequences of HpaII and Hin6I
cleavage fragments).
As with any microarray experiment, controls are
crucial for monitoring experimental variability. Suitable spiking DNA consists
of sequences that are frequently spotted on commercially available microarrays,
such as Arabidopsis, RNA spikes or
artificial sequences. (Note:
SNPs within the recognition sites for HpaII and Hin6I may simulate epigenetic
differences (8). In order to exclude the impact of DNA
sequence variation it is suggested to check the available SNP databases and
identify the DNA sequence variation within the restriction sites of the used
enzymes. From CpG island microarray studies, the estimate is that 10 to 30% of
methylation variation detected in between individuals could, in fact, be due to
DNA sequence variation (1). For comparison, in a study for the
Human Epigenome Project (HEP), interrogation of 3,273 unique CpG sites on
chromosome 6 revealed that 101 CpGs overlapped with known SNPs (3 %) (9). Another way to differentiate DNA sequence
effects from genuine epigenetic differences consists of performing an identical
microarray experiment on the same DNA sample that has been stripped of all
methylated cytosines (1). To eliminate all methylation, the
whole genome has to be amplified (thereby replacing all metC with C), for
example using the Phi29 DNA polymerase. Amplified DNA samples are then
subjected to the same steps as depicted in Figure 1 and co-hybridized on the
microarrays. In this experiment, all of the outliers must be a result of DNA
sequence variations within the restriction sites of the enzymes used and can be
eliminated from further epigenetic analyses).
- 400 ng of
genomic DNA is digested with 10 U HpaII plus 10 U Hin6I for 6 hours at
37°C. The DNA should be highly concentrated to keep the total volume of
the reaction below 20 µl.
- The samples
are then incubated for 10 min at
65°C to inactivate
the enzymes. Let the sample cool
down with 1°C/min. This step ensures reannealing of small DNA fragments
that can denature during heat inactivation.
- Immediately
proceed with the ligation reaction. It is important that the digested DNA
is processed as soon as possible to avoid degradation of the CpG
overhangs.
3.3 Ligation
- Take 200 ng of the digested DNA for a standard ligation. If
digested DNA is stored too long before ligation, ligation efficiency will
decrease.
- Adjust the buffer concentration to 1x, taking into account that the
unpurified restriction product still contains salts from the previous DNA
cleavage reaction. That means that buffer has to be supplemented for only
the extra volume added to the DNA.
- Add ATP to a final concentration of 1 mM. Add 120
pmol of the double-stranded CG-adapter and mix. (Note: The amount of CG adapter is directly dependent
on the amount of restriction enzymes used. For each enzyme add ~0.3 pmol
of the double-stranded CG-adapter per ng DNA. For instance, in a triple
digest (HpaII/Hin6I/AciI) of 500 ng DNA it is advised to add 500 x 0.3
pmol x 3 = 450 pmol of the adapter). Adjust the volume of the reaction with water to
18 µl, mix and then heat the sample for 10 min at 45°C in a water bath or
thermal cycler (Note: All ligation components have
initially combined without the T4-Ligase. Since the CpG-overhang of the
adapter is complementary to itself, it could form adapter-dimers during the ligation. Therefore, a heating step
is introduced before the ligase is added to the mixture. In this way,
adapters will be dissociated to bind specifically to the DNA overhangs
produced by the restriction enzymes).
- Take the sample out of the cycler and chill on
ice immediately. Add quickly 2 µl (10 U) of the ligase to a final volume
of 20 µl, mix well without introducing bubbles and place the tubes back
into the cycler. Incubate for 16 hours, followed by a short 5 min
inactivation step at 65°C. To favour re-annealing of short denatured DNA
fragments, let the sample cool
down with 1°C/min until room temperature is reached (Note: It is important to reduce the amount of ATP in
the mixture, since ATP interferes with the following McrBC digestion by
competing with GTP for a binding site in McrBC. Since ATP cannot be
utilized by the enzyme to perform the enzymatic reaction, McrBC will bind
to the DNA without cleavage. The best way to decrease the amount of ATP is
heat degradation, since free ATP is highly unstable).
- Store the ligation mixture at –20°C (4°C if the reaction is
processed at the same day).
3.4 McrBC digestion
- Perform the
reaction with NEB Buffer 2 + 1x BSA [10x] + 2x GTP [2 mM].
- Add buffer
only for the extra volume of the reaction. The ligation took already place
in a restriction enzyme buffer.
- ATP inhibits the reaction, therefore make sure that the DNA sample
was either purified before McrBC cleavage, or the present ATP was degraded
by heating the sample for some time during the “cool down” phase of the
ligation procedure. Digest the DNA with 10 U/µg of McrBC for 8 hours at
37°C (Note: McrBC makes one cut between each pair of
half-sites, cutting close to one half-site or the other, but cleavage
positions are distributed over several base pairs approximately 30 base
pairs from the methylated base. Therefore, the enzyme does not produce
defined DNA ends upon cleavage. Also, when multiple McrBC half-sites are
present in DNA (as is the case with cytosine-methylated genomic DNA) the
flexible nature of the recognition sequence results in an overlap of
sites, and so a smeared rather than a sharp banding pattern is produced.
GTP is more labile than most other nucleotides, so it is recommended to
aliquot the 100 mM solution supplied. McrBC will cut the DNA (both
strands) even if the methylated cytosines are on only one strand
(hemi-methylated).
- Heat-inactivate
the enzyme for 20 min at 65°C.
Store the ligation mixture at –20°C (4°C if the reaction is processed at
the same day).
3.5 CpG-specific adapter amplification
The protocol is based on
aminoallyl (aa) nucleotide
incorporation followed by coupling to N-hydroxysuccinimide (NHS) functionalized
dyes. This indirect labelling method is advantageous compared to direct
incorporation of dye labelled nucleotides in gDNA. The aminoallyl-nucleotide is
better tolerated by the Taq polymerase compared to other fluorescent
nucleotides analogs (Note: This protocol works only with standard Taq polymerases. Other,
high-processing polymerases usually can’t incorporate modified nucleotides
efficiently enough to produce PCR fragments of the desired length.). While direct
incorporation of dye labelled nucleotide protocols typically have incorporation
efficiencies in the range of 2-5 dye molecules per 1,000 nucleotides, the
aminoallyl protocol can achieve frequencies of incorporation in the range of
10-20 dye molecules per 1,000 nucleotides. Furthermore, the reagent costs for
the aminoallyl-NHS protocol are cheaper. Amino allyl-dUTP
contains a reactive amino group on a 2-carbon spacer attached to the methyl
group on the base portion of dUTP. During the
coupling reaction, after DNA synthesis, this amino group reacts with the NHS
ester of the monoreactive Cy3 and Cy5 dyes.
- For each 200
ng DNA, prepare the PCR-Mixture as follows (final volume 100 µl):
|
1st BS-PCR

|
Component
|
µl
|
(Amount) & [Conc.]
|
Comment
|
|
H2O
|
x
|
|
|
|
Buffer
|
10
|
Contains
15 mM MgCl2
|
|
|
MgCl2
|
14
|
[25 mM] à3.5 mM MgCl2
|
|
|
Allyl-dNTPs
|
0.5
|
250 µM
|
[20 mM]
|
|
Primer 1
|
1
|
~1pmol for each ng DNA
|
[200 pmol] Primer CG1b
|
|
Primer 2
|
0
|
Not
necessary
|
|
|
Polymerase
|
3
|
[5U/µl]
|
15 U
(depending on amount of starting DNA)
|
|
Template
|
x
|
200
ng
|
Adapter-ligation
product
|
|
Enhancer
|
0
|
|
|
|
Total
|
20
|
|
|
|
|
|
|
|
2.
Depending on the
Thermalcycler, the PCR reaction may have to be split into several tubes. Run
the following program:
|

|
Type
|
Step
#
|
Time
|
Temp
|
Cycles
|
Notes
|
|
1st PCR
|
þ
|
1
|
5:00 min
|
72°C
|
1x
|
Fills in 3’ recessed ends
|
|
2nd PCR
|
|
2
|
1:00 min
|
95°C
|
1x
|
Starting Denaturation
|
|
BS-PCR
|
|
3
|
0:30 min
|
93°C
|
Q
24x
|
Denaturing
|
|
TD-PCR
|
|
4
|
2:00 min +
3 sec/cycle
|
68°C
|
Annealing & Extension
|
|
Notes:
|
5
|
5 min
|
72°C
|
1x
|
Final Extension
|
|
6
|
hold
|
4°C
|
-
|
Storage
|
- Check the size of the amplified DNA by separating
8 µl product on a 1% agarose gel (the size of DNA should range from
approximately 150 to 2000 bp; see Fig. 3).

Fig. 3: The adapter-amplification results in a typical
smear in the size-range of 150 – 2,000 bp. To some
extent, the lengths of the amplified fragments depend on the primer annealing
temperature of the PCR reaction. Usually, an increased annealing/elongation
temperature will produce larger DNA fragments.
3.6 Purification of the amplification product
The overall yield of the
amplicon-PCR is relatively high (20-100 µg). However, most of the commercially
available column-based PCR cleanup kits cannot handle this large amount of DNA
without clogging. Therefore, our laboratory uses YM-50 Microcon columns
(Millipore) for cleanup, but other systems may work as well (Note:
Centrifugation of Microcon YM-50 columns forces liquid through the low-binding
cellulose membrane. Solutes larger than the nominal cut-off of the units are
retained: YM-50 columns have a double-stranded nucleotide cut-off of 100 bp. This includes about 46 bp
from the adapters on both sides of the amplification products. Therefore, all
methylation-sensitive restriction products below ~ 54 bp
and unincorporated adapters and primers should be removed during cleanup.
Recoveries of amplification products are generally above 90 percent. Millipore
columns are also available as plates, which could be suitable for
high-throughput analysis. Recently, we also used Qiagen
columns for purification; the overall amount that can be loaded is lower,
however the cleanup will still produce enough material for several
hybridizations with high quality DNA.).
- Add 500 µl nuclease-free water
to the empty columns and spin ~6 minutes at 10,000 rpm until the
column is dry. (The columns contain small amounts of Tris
on their membrane that can inhibit the subsequent labelling reaction.
Therefore, the columns have to be washed prior to adding the allyl-PCR
product).
- Insert the Microcon sample reservoir into a 1.5
ml vial. Pipette the whole PCR-product into the reservoir. Spin per guidelines
(approx. 1 min at 10,000 rpm). The exact spin-time is dependent on
temperature and rotor and may have to be elucidated empirically. Be sure
to align the strap of the collection tube cap towards the centre of the
rotor. Following the spin, the membrane should still be slightly wet.
- Add 500 µl dd-H2O and spin again 6
minutes at 10,000 rpm. The membrane should be looking wet, but should not
contain a visible amount of liquid. If there is liquid, spin it down for
another minute.
- Add additional 90 µl nuclease
free dd-H2O (Note: The following
aminoallyl/N-hydroxysuccinimide reaction requires that no free amines be
present in the coupling reaction buffer. This means that no Tris buffer can be used during any steps, including
the DNA-preparation.).
- Place the vial with the column into the Eppendorf
shaker and shake at 400 rpm for 1 minute (this helps to elute the entire
DNA which is maybe bound to the membrane). If you don’t have an Eppendorf
shaker, vortex the column slightly for several seconds.
- Uncap the Microcon unit. Separate the sample
reservoir from the filtrate cup, and place the sample reservoir upside
down into a new vial. Spin for 3 minutes at 4000 rpm in invert spin mode
to elute DNA.
- Remove the column. Cap the vial with the eluate
(~100 µl) for storage at –20°C.
- Use 5 µl of the eluate to measure the DNA
concentration in a spectrophotometer.
3.7 Labelling
3.7.1
Coupling
The amount of unmethylated DNA fraction to be hybridized to an array
depends on the surface area of the microarray. In our experience, 2 µg of the
eluate is enough for a standard slide.
- For all steps: Shield all samples, which contain
fluorescent-dyes from light!
- Dry your DNA sample (2 µg) in a speedvac.
- Resuspend the DNA pellet in 9 µl of the 0.1 M
Sodium-Bicarbonate Buffer. Add 3 µl DMSO. Let sample sit for 10-15
minutes.
- Denature the sample for 2 min at 100°C. In our
experience, single stranded DNA incorporates the Cy-dyes better compared
to native DNA.
- Quickly spin down and add the sample to a dry
aliquot of monofunctional Cy3 or Cy5 dye. Resuspend the dye pellets by
pipetting carefully; try not to introduce air bubbles (if it happens,
centrifuge the sample for a few seconds, this will get rid of the
bubbles).
- Incubate for 2 hours at 30°C in the dark.
3.7.2
Quenching
After the coupling step, reactive groups on the dyes must be quenched to
prevent cross reactivity or exchange of dye molecules between active aminoallyl
nucleotides. This protocol uses hydroxylamine to quench.
- Add 4.5 µl of 4 M hydroxylamine. Mix well by
pipetting up and down.
- Incubate for 15 minutes at RT (Eppendorf shaker,
400 rpm) in the dark.
- Combine the Cy3 and C5
samples.
3.7.3
Cleanup of product
To remove unincorporated dyes, columns have to be
applied that efficiently purify fragments in the size range of ~ 50 –
2,000 bp. For this protocol, we are using the Qiagen MiniElute PCR-purification kit.
- Add 25 µl of dd-H2O to the sample.
- Add 3 µl of 3 M NaOAc
(pH 5.2) to ensure a low pH of the mixture. It is important to keep in
mind the DNA binding curve for silica, on which this kit is based, is
favourable at low pH but falls off precipitously around pH 8. Thus it is
essential that the pH of your reaction be below pH 7.5.
- Add 275 µl (5x Vol.) of Buffer PB and mix. Apply
to column and centrifuge at 13,000 rpm for 1 min.
- Discard the flow-through and wash the DNA five
times with 750 µl Buffer PE at 13,000 rpm for 1 min. A critical step for
minimizing background is to effectively wash away all unbound dye
molecules during the final cleanup; small amounts of unincorporated dye
can have a large absorbance and thus give misleading results. If the
colour from the dyes is still visible in the wash, continue running wash
buffer through the column until the eluate is clear.
- Centrifuge the column for an additional 1 min at
maximum speed.
- Place the column in a new, clean 1.5 ml microcentrifuge tube and elute by adding 25 µl prewarmed (~50°C) elution buffer (EB) to the centre of
the membrane. Let it stand for 1 min at RT and spin at 13,000 rpm for 1
min.
- Repeat the elution step with an additional 25 µl
(final volume 50 µl).
3.7.4
Determination of incorporated dyes
- Place the entire 50 µl sample in a clean microcuvette. Measure the absorbance of the Cy3/Cy5
sample in a spectrophotometer at 260 nm (DNA), 550 nm (Cy3 fluorescence)
and 650 nm (Cy5 fluorescence). Be very careful about contamination in the cuvette as you will be recovering the sample for
hybridization. Wash carefully after each measurement.
- Calculate the amount of recovered DNA: A260 x 50
x total volume of sample (µl) = ng of target.
- Calculate the frequency of incorporation (FOI):
For Cy3 incorporation 86.5 x (A550/A260) and for Cy5 incorporation 51.9 x
(A650/A260). The results are expressed as the number of Cy-dCTP incorporated per 1.000 nucleotides of DNA (Note: The numbers 86.5 and 51.9 are conversion factors
calculated by using the average molecular weight of dNTPs (324.5 g/mole),
the absorption coefficient of Cy3 and Cy5 (150,000/M*cm and 250,000/M*cm)
and the concentration of Cy-labelled allyl-PCR products that absorbs 1 AU
of 260-nm light (50 µg/mL). When measuring ss-DNA, the concentration of Cy-labelled ss-DNA that absorbs 1 AU of 260-nm light is 37 µg/mL. The conversion factors changes then to 117 for Cy3
and 70.2 for Cy5. Note that sample volume is not factored in; this is
because volumes cancel out when DNA and cyanine absorbance are measured
simultaneously. Frequently, the FOI for the different dyes may differ. In
our experience, Cy5 is not as well incorporated as Cy3, however, it really
depends on several factors (humidity, ozone concentration, etc) and that
is why it is recommended to do reciprocal labelling). Optimal
frequencies of incorporation are between 15-20
and higher, though anything higher than 10 will give satisfactory results.
Using targets with less than FOI 6 may give high background or very weak
signals.
3.8 Hybridization
Before starting to hybridize,
it is recommended that the cover slips are already cut, the hybridization
chambers ready, and that all solution are prepared and
adjusted to the correct temperature. Also, scan one array prior to
hybridization to determine basal level of background on the array (Note:
As arrays age, some surface chemistries, particularly
poly-L-lysine, show an increase in auto-fluorescence in the Cy3 channel. Aged
microarrays may also show a higher affinity for salt, which will fluoresce in
the Cy3 channel. To minimize these effects use the arrays as
fresh as possible and store them properly (in the dark and under vacuum, if
possible).
3.8.1 Preybridization
- Briefly heat the Prehyb solution in an amber
reaction tube to 72°C and let it cool down to RT.
- To locate the area on the microarray that has to
be hybridized, place the dry microarray on a hybridisation template that
shows the dimensions of the slide and the area where the grids are
printed.
- Add water into the hybridization chamber to
prevent drying of the samples.
- Pipette the Prehyb-solution to the designated
area of the slide. Avoid surface contact with objects such as pipette
tips. If any bubbles appear, try to remove these with a syringe needle.
- When bubble-free, apply the cover slip carefully
(use cover slips larger then the grid-area). Use tweezers.
- Put the slide into a hybridization chamber and
pre-hybridize the array for 1 hour at 45°C.
- After incubation, move the slides to a 45°C jar
containing dd-water and remove the cover slip
carefully, without using force.
- Transfer the array into a new jar containing 45°C
dd-water and leave them there for 2 minutes,
moving the slide up and down several times. Repeat wash step 2 times.
- Transfer the slide again into a new jar
containing RT isopropanol and repeat the washing by moving the slide up
and down several times (~1 min).
- Immediately blow-dry the slide with pressurized
air. The pre-hybridized arrays can now be hybridized or stored in a dark
place for later usage.
3.8.2
Hybridization
- Place the complete 50 µl DNA sample in a speedvac (shield from light) until the solution is
reduced to about 5 µl. Do not allow the labelled DNA to dry completely!
- Preheat the Hyb-solution
prior to use for 5 minutes at 72°C. Make sure the Hyb-solution
is thoroughly resuspended. Centrifuge for 1
minute at 13,000 rpm to get rid of particulate material. Pipette the
needed amount (~60 µl for a 12,200 feature CpG island array) of the Hyb-solution and keep it at RT for 10 min.
- Add your DNA (~5 µl) to the Hyb-Mix,
denature it for 5 minutes at 80°C and briefly spin down.
- Add water into the hybridization chamber to
prevent drying of the samples.
- Pipette the Hyb-solution
to the designated area of the slide.
- Apply the cover slip carefully to the bubble-free
Hyb-solution. Again, avoid any contact with the
array-surface.
- Place the slide quickly, but carefully, into a
hybridization chamber to prevent renaturation of
the probe and evaporation of the Hyb-solution.
Make certain that the array surface is level.
- Protect the Hyb-chambers
from light, and hybridize for approx. 12-16 h at 50°C. (Oligo arrays may
require a lower temperature, whereas higher temperatures are needed for
BAC/PAC arrays).
3.8.3
Washing
- Fill three jars with Wash I and two
jars with Wash II and heat all to 50°C.
- Remove the cover slip by rinsing the slide
carefully in Wash I (Jar 2).
Take extreme care when handling the array. Allow cover slip to float off
the surface. Do not attempt to force off the cover slip as this will
likely damage the array.
- Place the array slide quickly into the second Wash I (Jar 2) for
15 minutes (all wash steps in the dark), moving the slide up and down from
time to time.
- Transfer the slides to a new jar (Jar 3) and
repeat the wash step for 15 min.
- Place the array slide into Wash II (Jar 4) for 15
minutes, moving the slide up and down from time to time.
- Transfer the slides to a new Wash II (Jar 5) and
repeat the wash step (15 min).
- Rinse the slide for 2 minutes
within another jar in dd-water (Jar 6) to remove
all traces of SDS and SSC. Transfer the slide into a new jar with
isopropanol (Jar 7) for 30 seconds, moving it up and down (Note:
Salt from the final wash buffer may precipitate out of solution onto the
array surface upon submersion into isopropanol. This is likely a result of
a higher than intended concentration of salt in the wash-buffer;
accidentally switching the wash buffers can cause this phenomenon. A brief
re-wash in water followed by another isopropanol wash will likely remove
the salt from the array).
- Blow the slide dry as quickly as possible using
pressurized air. Any buffer left on the array will appear as haze on array
and result in a strong green background.
- Scan the array in the Cy3 and Cy5 channel (532 nm
and 635 nm).
References
1. Schumacher, A., Kapranov, P., Kaminsky, Z.,
Flanagan, J., Assadzadeh, A., Yau,
P., Virtanen, C., Winegarden, N., Cheng, J., Gingeras, T., and Petronis, A. (2006) Microarray-based DNA
methylation profiling: technology and applications. Nucleic Acids Res 34,
528-542.
2. Cheng, J., Kapranov,
P., Drenkow, J., Dike, S., Brubaker, S., Patel, S.,
Long, J., Stern, D., Tammana, H., Helt,
G., Sementchenko, V., Piccolboni,
A., Bekiranov, S., Bailey, D. K., Ganesh,
M., Ghosh, S., Bell, I., Gerhard, D. S., and Gingeras, T. R. (2005) Transcriptional maps of 10 human
chromosomes at 5-nucleotide resolution. Science
308, 1149-1154.
3. Schumacher, A., and Petronis, A. (2006)
Epigenetics of complex disease: from general theory to laboratory praxis. Curr Top Microbiol Immunol 310,
81-115.
4. Schumacher, A. (2001) Mechanisms and
brain specific consequences of genomic imprinting in Prader-Willi and Angelman
syndromes. Gene Funct.
Dis. 1, 7-25.
5. Schumacher, A., Arnhold,
S., Addicks, K., and Doerfler,
W. (2003) Staurosporine is a potent activator of neuronal, glial,
and "CNS stem cell-like" neurosphere
differentiation in murine embryonic stem cells. Mol Cell Neurosci
23, 669-680.
6. Yamamoto, F., and Yamamoto, M. (2004) A
DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic
and epigenetic analyses. Mol Genet
Genomics 271, 678-686.
7. Li, J., Protopopov,
A., Wang, F., Senchenko, V., Petushkov,
V., Vorontsova, O., Petrenko,
L., Zabarovska, V., Muravenko,
O., Braga, E., Kisselev, L., Lerman,
M. I., Kashuba, V., Klein, G., Ernberg,
I., Wahlestedt, C., and Zabarovsky,
E. R. (2002) NotI subtraction and NotI-specific
microarrays to detect copy number and methylation changes in whole genomes. Proc Natl Acad Sci U S A
99, 10724-10729.
8. Schumacher, A., Friedrich, P., Schmid, J., Ibach, B., Eisele, T., Laws, S. M., Foerstl,
H., Kurz, A., and Riemenschneider, M. (2006) No
association of chromatin-modifying protein 2B with sporadic frontotemporal
dementia. Neurobiol Aging, Sept 14, epub
9. Rakyan, V.
K., Hildmann, T., Novik, K.
L., Lewin, J., Tost, J.,
Cox, A. V., Andrews, T. D., Howe, K. L., Otto, T., Olek,
A., Fischer, J., Gut, I. G., Berlin, K., and Beck, S. (2004) DNA methylation
profiling of the human major histocompatibility complex: a pilot study for the
human epigenome project. PLoS Biol 2, e405.