UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Author: B. Griffith
Revision level: 5
Effective Date: 1/06/2005
Primary Reviewers: B Griffith
1. PURPOSE
This protocol describes the setup of the Genomic Solutions GeneTac hybridization unit
and the hybridization of Cy3 and Cy5 labeled cDNA targets to the 70mer probes printed
on microarray slides. As an overview, we combine equal numbers of pmoles of labeled
Cy3 and Cy5 targets, heat denature the targets and apply the targets to slides waiting in
the programmed and prepared GeneTac unit. After the appropriate hybridization
incubation time, the GeneTac unit performs the washes and the staff overseeing the unit
performs a final wash and rapid drying of the hyb’d slides in preparation for scanning.
2. SCOPE
This protocol has been implemented in the EBL under the direction of Drs. Lyons and
Williams. Generally, we hybridize PLL slides produced and printed with 70mer
oligonucletide probes in our lab. We found that hybridizations at 42C in 30% formamide
overnight, give excellent signal and maintain the stability of the PLL coating.
3. MATERIALS
3.1. 20X SSC pH 7.0
NaCl (JT Baker Cat # 4058-05, Ultrapure)
NaCitrate (Sigma Cat# S4641 ACS grade)
3.2. salmon sperm DNA (Sigma Cat# D7656, phenol/chloroform extracted, 10.5 mg/ml)
Store at –20C
3.3. SDS (Sigma Cat# L4390, 99% purity, Na salt, Molecular Biology grade)
3.4. Formamide (Sigma Cat# F9037, deionized, 99.5% purity)
Store at 4C
3.5. Sarkosyl (Eastman Kodak, IB07080, Molecular Biology Certified DNase and RNase
free)
3.6. gaskets (Genomic solutions Cat# HYB 10155 )
3.7. red o rings (Genomic solutions Cat# )
3.8. black metal slide holders (Genomic solutions Cat# H2000178)
3.9. amber slide covers (Genomic solutions Cat# H2000174)
3.10 probe plugs (Genomic solutions Cat# HYB10102)
4. REAGENT PREPARATION
4.1. 20X SSC pH 7.0
3M NaCl and 0.3M NaCitrate is the composition of 20XSSC
FW of NaCl = 58.44 gms/mole (JT Baker Cat # 4058-05, Ultrapure)
FW of NaCitrate = 294.1 gms/mole (Sigma Cat# S4641 ACS grade)
Dissolve 175.3 gms NaCl and 88.2 gms NaCitrate in approx 700 mls double
deionized water and adjust pH to 7.0 with concentrated HCl. Then bring into final
volume of 1 liter with double deionized water. Autoclave and store at room
temperature.
4.2. 10% SDS
Dissolve 10gms of SDS in 90mls of sterile double deionized water. Once dissolved,
bring final volume up to 100mls with additional sterile double deionized water. Do not
autoclave the 10% SDS.
Page 1 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Author: B. Griffith
Revision level: 5
Effective Date: 1/06/2005
Primary Reviewers: B Griffith
4.3. 5% Sarkosyl
Dissolve 5 gms of Sarkosyl in 90mls of sterile double deionized water. Once
dissolved, bring into final volume of 100mls with additional double deionized water.
4.4. 1X SSC/0.03% SDS (for 100mls wash #1)
Combine
94.7 ml ddH2O
5.0 ml 20X SSC
0.3 ml 10% SDS
Place 200 mls in the wash bottle #1 of the GeneTac
4.5. 0.2X SSC (for 100 mls of wash #2)
Combine
99 ml ddH2O
1 ml 20X SSC
Place 200 mls in the wash bottle #2 of the GeneTac
4.6. 0.05X SSC (for 100mls of wash #3)
Combine
99.75 ml ddH2O
0.25 ml 20X SSC
Place 200 mls in the wash bottle #3 of the GeneTac
5. PROCEDURE
5.1. Overview of procedure
5.1.1. Step 1: Set up the GeneTac hybridization unit
1. Perform valve maintenance and dry manifold lines before placing
slides onto unit
2. Prepare the slide holders with gaskets and put into hybridization
chambers into the unit
3. Prepare the wash buffers and place in unit’s bottles
4. Review the hyb software program and insert disk into unit
5. Initiate the program so the hyb chambers preheat to the loading
temperature
5.1.2.
Step 2: Prepare the targets for the hybridization
1. Make fluorescent targets in advance
2. Prepare hybridization solution
3. Combine Cy3 and Cy5 targets into single tube with hyb solution
4. Denature targets
5. Load targets into preheated hybridization chamber
6. Hybridize overnight
5.1.3.
Step 3: End of Hybridization
1. GeneTac completes automatic washes
2. Perform final manual wash
3. Spin pairs of slides dry
Page 2 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Author: B. Griffith
Revision level: 5
Effective Date: 1/06/2005
Primary Reviewers: B Griffith
4. Store in black, opaque box
5. Scan immediately
6. Perform maintenance in GeneTac
5.2. Step 1: Setting up the GeneTac Hybridization unit
5.2.1. Valve maintenance and manifold purging
1. Turn on the power to the GeneTac (rocker switch on the rear) without a
floppy disk in the drive. Push floppy disc into drive after GeneTac has
booted up.
2. Click on maintenance/valve
3. Manually perform “maintenance” to check valves. Individually open and
close every valve to be sure each one clicks open and clicks closed.
You don’t want to use a module if the valves are not opening and closing
properly. This step requires approximately 2 minutes to complete.
If valve does not click, indicating an opening or closing, see 5.3.2 at the
end of this procedure.
4. Return to first screen of valve maintenance and click on “air input,
wastebottle 1 and primary primer”. Let air purge through the top maniforl
for 1 min. Then click on “air input, wastebottle 1 and secondary primer”.
Let the air purge through the bottom manifold for 1 min. This is to assure
that no fluid inadvertently moves later, from the manifold to the slide
chambers. The air input line is separate from all the fluid lines, so the
fluid lines can either be resting in the wash buffer reservoirs or in the
water wash reservoir, during this manifold purging process.
5.2.2. Preparing slide holders and placing in GeneTac Hybridization unit
1. The slide holders are comprised of 2 parts: the lower support is a black
metal plate with a raised loop at the top end of the plate. The upper
cover is an amber plastic/plexiglass with grooves for gaskets and with
metal shims.
2. Always wear gloves while handling the slide holder components and the
slides, to prevent oils and smudges from getting onto the slide holders
and the slides.
3. The amber slide cover must never be abraided or scratched.
4. The metal shims must be perfectly flat. Use a small jeweler’s
screwdriver to loosen or tighten one of the 6 small screws nearest the
bow in the shim. Generally, I have had to loosen the screws by a partial
turn and then run my gloved finger along the shim toward the screw and
then retighten lightly.
5. Be sure that the 4 tiny red 0 rings at the “top” of the amber slide cover,
are seated in their grooves around the inlet/outlet ports.
6. Always use new black o-rings in the slide covers. Once the black 0-rings
are compressed, they can never be assured to seal tightly again. So,
you cannot pretest the seal of the gasket. With gloves on, place the
black o-rings into the larger grooves on the face of the cover plate. Do
not twist the black o-rings as the units will leak if the black o-rings are
twisted and not smooth. Use your gloved finger to slide the black o-ring
into the groove. It should lie flat.
7. Examine the black slide holder and be sure that the tiny prongs at the top
and bottom are slightly raised i.e. < or = 1 mm.
8. Use purified compressed nitrogen gas to dust the microarray slide
surfaces and the amber cover, taking care not to loosen the gaskets in
the amber cover. Dusting helps prevent bubbles and keep hyb solutions
even over the slides.
Page 3 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Revision level: 5
Effective Date: 1/06/2005
Author: B. Griffith
Primary Reviewers: B Griffith
9. Place the microarray slides, dna face up and label face down, on top of
the black metal slide holder. Be sure that the label is at the top of the
slide holder. Seat the two slides into the holder by tilting the top right
corner downward, so that the slides seat to the top right corners. Make
sure that the slide is laying flat in the holder. If the slide is sitting on a
prong and is not laying flat, the slide will crack when pressure is applied
in the next step
10. Place the amber plexiglass slide cover , gasket side down, ontop of the
slides being held in the black metal slide holder. . Listen for the sound of
glass crunching… reseat if you hear this sound. Hold firmly together and
place onto the GeneTac unit. Keep holding the unit together, with your
finger through the metal grid, while you tighten the metal grid down onto
the slide holder unit. Tighten the black screw knobs, finger tight. Do not
use tools to tighten. If you over tighten, you could break the slide cover
unit.
5.2.2 Prepare the wash buffers and place in GeneTac Bottles
1. The correct input lines must be placed in the wash buffer bottles. So the
line marked 1 is for the first wash, the line marked 2 is for the second
wash, and so forth.
2. The wash buffers with SDS must be clear. NEVER allow any buffers
containing particulates, enter the microfluidics. Precipitates, such as
SDS, can plug the fluidic lines which are difficult to clean (can take 8 hrs
of work to remove particulates!) Make 200 mls of each wash buffer.
3. If you are only using 3 different wash buffers, then input lines 4 and 5
can be left in the air or left in a water bottle reservoir.
4. Wash bottle #1: 1X SSC/0.03% SDS
5. Wash bottle #2: 0.2X SSC
6. Wash bottle #3: 0.05X SSC
7. Be sure that the two waste bottles are empty or low in volume and that
the lines from the vacuum port to the waste bottles are tight. This
assures the wash fluid movement.
5.2.3 Review the Hyb software program, turn on GeneTac, and insert floppy disk into
GeneTac unit
1. On a PC, review the Hyb software program. We use a 6 step program
entitled “42C 17 2 4 min washes.hyb” thus
a. Oring conditioning: Temp:75C Time: 2 min:00s Agitate: No
b. Introduce Probe: Temp: 42C
c. Hybridization: Temp:42C Time: 17 hr:00m:00s Agitate: Yes
d. 1x/0.03% SDS 2 cycles, Source 1, Waste 1 at 25C, Flow for 15s,
Hold for 45s
e. 0.2XSSC 2 cycles, Source 2, Waste 1 at 25C, Flow for 30s, Hold
for 1 min:30s
f. 0.05XSSC 2 cycles, Source 3, Waste 1 at 25C, Flow for 30s, Hold
for 1m:30s
2. Start the GeneTac hybridization program from a floppy disk. The programs
can be edited and reviewed with the software on the scanner’s PC and then
placed in the GeneTac to generate the run. The programs on the floppy
cannot be edited or reviewed once placed on the GeneTac.
3. Insert the floppy containing the program and select “start a run”. Touch the
name of the desired program and follow the steps to start including selection
Page 4 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Revision level: 5
Effective Date: 1/06/2005
Author: B. Griffith
Primary Reviewers: B Griffith
of the hyb stations (ie 1-12 or a subset), holding off to start the first
incubation /gasket seating step until you have the fluorescent target in the
95C heat block for 2 min. It takes less than 5 minutes for the gasket
seating/heating step to complete so you can be denaturing and pulsing down
your fluorescent target, while the gaskets are heat seating in the GeneTac.
5.3 Step 2: Prepare targets for the hybridization and start hybridizations
5.3.1 Prepare hybridization solution
1. For preparation of the hybridization solution for PLL slides being hybridized
overnight in 30% formamide, 5xSSC, 100ug/ml salmon sperm DNA, 1% sarkosyl,
0.1% SDS and 200 pmoles of dye in Cy3 target and 200 pmoles of dye in Cy5
target, see the excel worksheet entitled “Hyb Worksheet 30% formamide
Manual&GeneTac.xls”
2. Determine the number of hybs or slides being performed with the same batches
of Cy3 and Cy5 target. Place the number of hyb value into the excel
spreadsheet. Calculate the volume of target (Cy3 and Cy5) required to hyb each
slide with 200 pmoles of Cy3 dye in target and with 200 pmoles of Cy5 dye in
target. Insert these two volumes electronically into the excel worksheet and the
volume of water will be calculated.
3. To a single tube, add each of the reaction components, but be sure that you add
the detergents last! (SDS and Sarkyosyl) (keep wrapped in foil). If you add the
detergents to the high salt (20xSSC), the salt can cause the detergents to
precipitate out.
4. Heat denature at 95C for 2 min in heat block
5. Spin tubes down at 11,000 x g for 1 min. Hold target tube in foil at room temp
and immediately begin loading into hyb chambers. Note: Do NOT place on ice
as SDS will precipitate out.
5.3.2 Load targets into preheated hybridization units
1. When the GeneTac shows it is ready for the “Probe”, touch “probe” for
the slide position and remove the inlet peg from atop the side cover.
Touching ‘probe” opens the valve to the slide position, which allows for
the buffer to be loaded. GeneTac calls the cDNA probe and most call it
target
2. Use a P200, to load the 110ul of hyb buffer containing the target. Hold
the pipetter at 90 degrees, relative to the surface of the slanted amber
plexiglass slide cover. Put the tip of the pipettor as far into the hole, as
the pipettor will go. Slowly and continuously dispense the fluid into the
chamber. Do not express any air. Withdraw the pipettor and
immediately reseat the tiny white/clear plug into the loading hole.
3. Press “finish” on the keypad for that slide position. Continue to the
remaining slide positions.
4. Look for signs of leakage from the top of the slide or from the sides of the
slides. There is little that you can do if it starts to leak.
5. Look for signs of bubbles and note them. Generally the bubbles are very
tiny, they go away and don’t effect the hyb
6. Hybridize 17 hours (overnight)
5.3
Step 3: End of Hybridization
5.3.1 Final Washes
Page 5 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Revision level: 5
Effective Date: 1/06/2005
Author: B. Griffith
Primary Reviewers: B Griffith
1. Cathy Scott of Genomic Solutions recommended NOT using a drain cycle as
part of the hyb program as it may cause some unusual background signals.
2. After all the GeneTac washes are completed and the program I indicates
that it is finished, you can immediately and rapidly dismantle the slide unit,
carefully withdrawing the unit from the GeneTac. Remove the slide cover
and with gloves, remove the slides quickly to a black slide carrier with
handle. Immediately and quickly proceed to the water wash.
3. Fully rapidly plunge the slide up and down 3 x 10 times in ddH2O in a tray,
to try to remove dye that has stuck under/around the slide gasket. Keep the
slides under the water and use short up/down movements.
4. Then remove the slide rack from the water. Quickly pull out one slide and
tap hard on long edge onto kimwipes three times, turnover and tap opposite
long edge three times on kimwipes hard, and finally return to original long
edge and tap three more times onto kimwipes. Then return the slides to a
dry slide carrier, spin the slides in a slide holder carried on a microtiter plate
rotor at 700 rpm for 5 minutes to dry the slides. After centrifugation, place
the slides and a freshly dusted , black slide box. Keep the slides in the dark!
Extreme speed is absolutely necessary so that no salt solution dries on the
slide; dried salt gives a nasty punctuate green background.
5. Keep the slides in a dark box. Immediately scan them.
5.3.2 Clean-up/Maintenance
1. Reassemble the slide holder with two dummy, clean slides before proceeding
with the instrument maintenance. You can leave the hyb black gaskets in
place for this maintenance.
2. Place all 5 input lines into a bottle of ddH2O.
3. Use the touch pad to start the cleaning maintenance for the positions
containing slide units with slides. Water will be drawn through all the fluidics
and across the slides.
4. When the maintenance is complete, you can remove the slide unit and
dissemble.
5. Remove and discard the used black o-rings. They cannot be reused. You
can remove them by poking them carefully with a needle to pull them out.
6. The black slide holder and the amber slide cover can be soaked for a few
minutes in ddH2O with a little detergent, rinsed at least 3 times in ddwater
and then gently dried with kimwipes. Blow compressed air into the channels
and fluidics of the cover to dry them out, including the underside slots, the
top slot, the bottom slot, the gasket slot and the channels in the chamber.
5.2.3 Manual Flush
Jim Galt at Genomic Solutions suggested this procedure when one of the valves
was not opening/closing during the maintenance valve check.
1. Assemble the slide holder with two clean dummy slides.
2. Click on maintenance and then valve, from the main screen.
3. Open the valves for Bottle 1, Waste 1, and the 3 valves for the slide/position
that is not opening.
4. Take a syringe with a 20 gauge needle (yellow), and insert it into the Bottle
1 input line and push water through the system with the syringe, you should
be able to the water flow across the slide and into the waste bottle. If the
valve is stuck, there will be resistance to pushing the syringe. Quite a bit of
force may be needed to free the stuck valve, take care as use of excessive
force may cause the port plug to shoot out of the amber cover.
Page 6 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Author: B. Griffith
Revision level: 5
Effective Date: 1/06/2005
Primary Reviewers: B Griffith
Page 7 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Author: B. Griffith
Revision level: 5
Effective Date: 1/06/2005
Primary Reviewers: B Griffith
Hybridization Worksheet: PLL slides overnight manual or in GeneTac
Date:
Purpose of Experiment:
Targets used: Cy3:
Cy5:
Revised 3/24/2004
BG
Hyb buffer :
30% formamide,5xSSC, 0.1% SDS, 1% sarkyosyl, 100ug/ml ssDNA, 200pmoles each dye labeled target per slide
final concentrations
Manual Hybs
GeneTac Hybs
Stock conc and Reagent
20XSSC
ssDNA 10.5 mg/ml
cDNA Cy3 (200pmole of dye/hyb)
cDNA Cy5 (200pmole of dye/hyb)
Formamide
water
5% Sarkosyl
10%SDS
Total
# of hybs=
2
Vol ul
Vol/# rxn final Conc
13.75
27.5 5xSSC
0.55
1.1 100ug/ml
5.5
11 200pm/hyb
5.5
11 200pm/hyb
16.5
33
30%
3.3
22
1%
0.55
1.1
0.10%
55
110
# of hybs=
4
Vol ul/rxn Vol/# rxn final Conc
20XSSC
27.5
110 5xSSC
ssDNA 10.5 mg/ml
1.1
4.4 100ug/ml
cDNA Cy3
11
44 200pm/hyb
cDNA Cy5
11
44 200pm/hyb
formamide
33
132
30%
water
3.3
13.2
5% sarkosyl
22
88
10%SDS
1.1
4.4
0.10%
Total
110
440
Note: volume of cDNA's varies depending on fluorescent specific activity. Lyopholized targets can be resuspended
in appropriate volume. We use 200 pmoles per 110 ul of hyb solution on the GeneTac
Page 8 of 9
UNM SOM EXPERIMENTAL BIOTECHNOLOGY LABORATORY: MICROARRAYS
Standard Operating Procedure
Title: cDNA Hybridization protocol using GeneTac Hybridization Unit
SOP#: M 6.3a
Author: B. Griffith
Revision level: 5
Effective Date: 1/06/2005
Primary Reviewers: B Griffith
Hyb Temp:
42
Hyb Time:
overnight
Slide #'s Used:
Slide Print Date:
PLL slide production Date:
Other slides used:
Hyb oven used for manual hybs: Williams vac oven at 42 with pipette box humid chamber inside
Coverslips used: Fisher, water/ethanol washed manually
Notes on Hyb:
Page 9 of 9
42
overnight