Leiden Genome Technology Center (LGTC) - LUMC©
Labeling and hybridization of
genomic amplicons on CpG island
microarrays
Array protocols
(Peter-Bram 't Hoen and Judith Boer; last modified 8 February, 2005)
for in house use only
Abbreviations
Purpose
This protocol describes the random-primed aminoallyl labeling, Cy-dye coupling and
hybridization of genomic DNA to CpG island microarrays. This protocol can be
applied to linker-mediated PCR products (amplicons) for differential methylation
hybridization and ChIP-on-chip assays.
Protocol
BOOKING MACHINES AT THE LGTC
Before you start, go to the LGTC web site (http://www.LGTC.nl/) and book the hybridization
station modules and scanner time that you need on the Yahoo calendar. Also, fill in an order
form.
TIME SCHEDULE
Day 1 (full day): aminoallyl-labeling of amplicons, start hybridization overnight.
Day 2 (morning): wash and scan microarrays.
AMINOALLYL RANDOM-PRIMED LABELING (using BioPrime kit)
1. About 0.5 μg DNA is needed for the labeling in a maximum volume of 24 μl.
Speedvac sample to concentrate if necessary.
2. Mix at room temperature:
500 ng CpG amplicons/water
2.5x random primer (N8) solution
Total volume
24 μl
20 μl
44 μl
3. Denature at 95C in PCR block for 10 min, transfer to ice block for 1 min. Spin
briefly, then keep on ice.
4. Add on ice:
10x dNTP aminoallyl mix
5 μl
Klenow (BioPrime kit)
1 μl
Total volume
50 μl
5. Incubate at 37C for 2 hours or overnight (using water bath).
6. Add 5 μl stop mix (BioPrime kit) and 45 μl water to a total volume of 100 μl, keep at
RT.
7. Clean up with QIAquick PCR purification column (all spins are 1 min at 13K, RT):
 use 400 μl of PB buffer
 wash three times with 500 μl 75% EtOH (NOT WITH PE BUFFER!!!)
 spin dry
 elute with 2 x 50 μl alkaline water, pipetted to the center of the column, allow to sit
for 1 min
8. Take 1.5 μl aliquots for Nanodrop, use alkaline water as blanc. Expected yield is at
least 2-3 μg.
CY-DYE COUPLING
9. Take 1.5 (- 8) μg aminoallyl-modified DNA from random-primed labelling reaction.
10. Speedvac to ~ 3.3 μl (about 30 min at 60C). Do not let sample dry completely. If
necessary, bring volume to 3.3 μl with alkaline water.
11. Mix at RT:
1.5 - 8 μg aa-modified DNA in water
3.3 μl
5 μl
DMSO (dye box, 4C)
0.3 M sodium bicarbonate pH 9.5
1.66 μl
12. Transfer sample to a dry aliquot of Cy3 or Cy5 mono-reactive dye and resuspend by
pipetting.
13. Incubate 1 h at RT in the dark, flick tubes to mix every 15 min.
14. Add 4 μl 4 M hydroxylamine (total volume 14 μl).
15. Incubate 15 min at RT in the dark.
16. Perform YM-30 filter purification (all spins are at 13K, RT):
 prewet column with 100 l TE
spin 5 min
 add 186 μl TE to both Cy3 and Cy5 samples
transfer both samples to the same YM-30 column
spin 8 min
 discard flow-through
 add 500 μl TE and 10 l Cot-1 DNA
spin 8 min
 wash with 500 μl TE
spin 8 min
 add 500 μl TE, 2 μl tRNA and 2 μl poly A RNA
spin 8 min
 ONLY if retained volume is larger than 48 μl
spin 1-5 min extra
 put column upside down in new tube
spin 1 min
17. Add TE up to 48 μl and store at 4C in the dark until use.
18. Measure coupling efficiency of 1.5 μl aliquot on Nanodrop using microarray protocol.
At least 50 pmol of cy-labeled material is necessary for a good hybridization. Use all,
since otherwise the amount of blockers in hyb is less. Lower amount of input at step 9.

HYBRIDIZATION
19. Prewarm 75% Hybridization master mix for a few minutes at 37C.
20. To 48 μl Cy-DNA in TE, add 3 μl 10% SDS and 100.2 μl prewarmed 75%
Hybridisation mix (fume hood!). Take care with air bubbles, as this mix is extremely
viscous!
21. Incubate for 10 min at 70C (PCR block lab); don't spin.
22. Put at 37C for 1h (PCR block lab).
23. Assemble hybridisation module for GeneTAC hybstation:
 take microarrays from LGTC dessication cabinet
 please note that the microarrays are not marked!
 orient the slides so you can read the CodeLink text at the top
 align slides to the upper right corners of the black bottom unit
 when hybridizing an uneven number of slides, use a dummy
 place black rings in the plastic top unit
 carefully place the plastic top unit, make sure all red rings are present
 make sure the unit is aligned, otherwise slides will break
 place module on hyb station position and screw hand-tight
 place a white plug in the hole to close the module
24. Load hybridization program 24HRS37C and start when ready:
a. module is prewarmed for 5 min at 37C
b. when prompted by the hybstation ('probe'), pipet hybridization solution in
module, using the touch screen to break vaccuum
c. the fluid should travel all the way to the top of the slide without airbubbles
d. replace plug and press 'finish': slides are hybridized at 37C
e. install wash solutions 1-5 (see below) using tubes 1-5 on the hybstation,
making sure there is no precipitate or growth in the bottles
f. stop the hybridization after 14-16 h, one module at a time
g. start the wash protocol, per module: PBFWASH (takes about 15 min)
25. PBFWASH protocol (20 sec flow; 40 sec hold) solutions and temperatures:
1
2
3
4
Wash solutions
50% formamide / 2x SSC
PN buffer
0.2 x SSC
0.1 x SSC
Temperature
35C
25C
25C
25C
5
0.01 x SSC
25C
26. Prepare fresh baths with alcohol dehydration series: 70%, 90%, 100% EtOH (note: a
bit of water in the 100% EtOH will leave ugly spots on your array!).
27. As soon as the wash protocol is finished, take slides out and dehydrate by 1 min
incubations in 70%, 90%, 100% EtOH. Keep dark as much as possible.
28. Please note: the slides are still not marked, so make sure to know who is who.
29. Place slides top-up in a tube rack and blow dry with compressed air in fumehood.
30. When visible dried-in drops remain, repeat EtOH series and drying.
31. Scan slides in Agilent scanner at 10 μm resolution. Tiff split UL to LR. Burn original
scan and split tiffs to a CD.
32. When slides appear not washed well, repeat the last three washes by hand, dipping
only very briefly in the 0.01 x SSC, and rescan. Mark and store slides at RT in dark.
33. Clean up the hybstation and modules:
a. place used modules with dummy slides on the hybstation
b. run cleaning protocol
c. place plastic tops, plugs and black rings in milliQ water
d. prepare a beaker with 500 ml 0.1 M HCl (from 1 M HCl stock)
e. take one module and work quickly: flush all 4 channels three times using a
p200, then transfer to fresh milliQ, repeating the flushing of the channels
f. blow dry, but make sure the red rings don't get lost
g. incubate black rings and white plugs for 2 min in just-boiled (90C), dry
Material

BioPrime random labeling kit (Klenow, N8 solution, stop solution): Invitrogen

dNTP mix for aminoallyl labeling (50 ul):
amount stock
1 ul 100 mM dATP (Fermentas)
1 ul 100 mM dCTP (Fermentas)
1 ul 100 mM dGTP (Fermentas)
1.75 ul 10 mM dTTP (Fermentas)
 2.4 ul 50 mM aa-dUTP 5-(3aminoallyl)-dUTP (Ambion)
final concentration
2 mM
2 mM
2 mM
0.35 mM
2.4 mM

QIAquick PCR purification columns (Qiagen) Use fresh kit!

alkaline water: 1 ml milliQ + 15 ul 0.3 M sodium bicarbonate pH 9.0

DMSO: 27685-5, Aldrich

Na-carbonate buffer: 0.3 M bicarbonbate; adjust pH to 9.0 with NaOH, store in small
aliquots at -20ºC to preserve pH.

hydroxylamine: 4.0 M in water (Sigma), store at -20˚C in dark.

Cy3 and Cy5 dyes: mono-reactive (PA23001 and PA25001, Amersham):
1. work quickly and protect from light as much as possible
2. dissolve one vial of monoreactive dye in 20 µl dry DMSO
NOTE: do NOT use DMSO from an old bottle (replace every six months; store
dry with silica); remove DMSO with a needle and syringe through the septum.
3. aliquot immediately into 20 portions of 2 µl and speedvac till dry (the LGTC
has a speedvac with vapor trap, 30 minutes at 50˚C)
4. store aliquots at 4ºC in the presence of some silica

TE: 10 mM Tris.HCl, 1 mM EDTA (pH8.0)

Microcon YM-30 columns (Millipore)

ethanol: 100%, 90% and 70% for dehydration series, 75% for QIAquick washes

10 % (w/v) sodiumdodecyl sulfate (SDS)

polyA+ RNA: 10 µg/µl (4ºC; P9403, Sigma)

yeast tRNA: 10 µg/µl (4ºC; R8508, Sigma)

Cot1 DNA: 1 µg/µl (-20ºC; 15279-011, Invitrogen)

75% Hybridization master mix:
1g dextran sulphate (USB, US 70796 )
5.3 ml formamide 100% (Invitrogen, 15 515-026 )
0.7 ml MilliQ H2O
1.0 ml 20x SSC (Sigma, S6639)
pH 7.0

50% formamide/2x SSC (500 ml): to 250 ml 100% formamide (Fluka, 47670), add
200 ml milliQ water and 50 ml 20x SSC, mix.

PN-buffer: 0.1 M Na2HPO4 /0.1 M NaH2PO4, pH 8, 0.1% Igepal CA630 (Sigma,
I7771); filter through 0.22 m filter.

0.2x SSC, 0.1x SSC, 0.01x SSC: dilute from 20x SSC stock in milliQ water; filter
through 0.22 m filter.
| Top of page | LGTC homepage |
| Human & Clinical Genetics |