Microarray Protocols
Scientists at the OMRF Microarray Research Facility are available to address technical
questions about cDNA production, labeling, and hybridizations. Many parameters have
been tested and optimized for the particular equipment in our facility. We request that
you cite our facility in publications if you find these protocols useful. The following
protocols are available for downloading.
Protocols
Succinic Anhydride Slide Blocking Protocol
Direct Cy-Labeling Protocol with Omniscript RT (modified for Ventana Discovery)
Direct Cy-Labeling Protocol with EndoFree RT (modified for Ventana Discovery)
Indirect Cy-Labeling Protocol (OMRF Modified Amersham CyScribe Labeling)
Automated Hybridization Protocol (Ventana Discovery Optimized Protocol)
Validation
Omniscript cDNA Synthesis for Quantitative Real-Time PCR (QRT PCR)
SYBR Green I Quantitative Real Time PCR (QRT PCR) Protocol
Quantitative Real-Time PCR (QRT PCR) Using EndoFree Reverse Transcriptase
Succinic Anhydride Slide Blocking Protocol
OMRF oligonucleotide microarrays are UV-crosslinked but not blocked. They may be
blocked individually or as many as 24 blocked at a time.
Materials Needed for a small number of microarrays:
50 ml conical polypropylene tubes with caps (various vendors)
Succinic Anhydride (Aldrich #23969-0 or Sigma #S7626)
1-methyl-2-pyrrolidinone (Aldrich #270458)
Boric Acid (various vendors)
Sodium Hydroxide (various vendors)
0.2  Filtered MilliQ or Nanopure Water
95% Ethanol (various vendors)
Orbital Shaker
Rack for 50 ml tubes
Hemostat or Tweezers
Centrifuge for 50 ml tubes or compressed nitrogen gas cylinder
0.2  Filter & Bottle
Diamond tip pen
Materials Needed for a large number of microarrays:
Slide staining rack or tray
Succinic Anhydride (Aldrich #23969-0 or Sigma #S7626)
1-methyl-2-pyrrolidinone (Aldrich #270458)
Boric Acid (various vendors)
Sodium Hydroxide (various vendors)
0.2 micron Filtered MilliQ or Nanopure Water
95% Ethanol (various vendors)
Orbital Shaker
Compressed nitrogen gas cylinder
0.2  Filter & Bottle
Blocking Procedure for Individual or a Few Slides:
1. Prepare 0.2 M sodium borate (pH 8.0) by dissolving boric acid in water and
adjusting the pH to 8.0 with NaOH. Filter sterilize. Store at room temperature.
2. Mark array area using the diamond tip pen by etching the back of the slide to
serve later as a guide for the cover slip. Place slides in a container or box for
easy access.
3. Working in the fume hood, transfer 31.5 ml of 1-methyl-2-pyrrolidinone to a 50 ml
conical tube.
4. Weigh out 0.5 g succinic anhydride and bring it to the fume hood.
5. Add the succinic anhydride to the 1-methyl-2-pyrrolidinone in the 50 ml tube. Cap
the tube and gently mix until the flakes dissolve (this takes about 1 minute).
6. Just after the last flak e of succinic anhydride dissolves, add 3.5 ml of 0.2 M
sodium borate (pH 8.0) Cap the tube as soon as possible and mix once by
inverting.
7. Remove the cap and immediately submerge a slide (or two slides back to back)
into the solution. Recap, tap the tube firmly to dislodge any air bubbles attached
to the glass, and begin agitation by transferring tubes to a rack on an orbital
shaker so that the slides are horizontal and will remain fully submerged. Shake
gently for 15 minutes (we use setting 2.5 on our Hoefer "Red-Rotor" shaker that
goes from settings 0 to 10).
8. After 15 minutes, use a hemostat or tweezers to transfer each slide to a separate
50 ml tube with ~ 45 mL room temperature water. Cap and agitate on the orbital
shaker in the horizontal position for 2 minutes.
9. Transfer each slide to a separate 50 ml tube with ~ 35 ml of 95% ethanol, cap,
and gently invert for about 5-10 seconds.
10. Transfer the slides to an empty, clean, dry 50 ml centrifuge tube and cap. Dry the
slides by centrifuging them for 5 minutes at ~1000 rpm. Immediately remove
slides from tubes at the end of the spin, allow a few seconds for any residual
moisture to evaporate, and transfer slide to a clean container for storage.
11. Store slides dry at room temperature until hybridization. The microarrays are now
ready for hybridization.
High Throughput Blocking Procedure: We follow the Corning CMT-GAPS II
manual, soaking up to 24 slides in a rack and slide staining dish, or in a beaker with
the succinic anhydride/ sodium borate solution.
1. Prepare 0.2 M NaBorate (pH 8.0) by dissolving boric acid in water and adjusting
the pH to 8.0 with NaOH. Filter sterilize. Store at room temperature.
2. Dissolve 4.0 g succinic anhydride in 252 ml 1-methyl-2-pyrrolidinone in 500 ml
beaker.
3. Put slides in slide holding rack.
4. Just after the last flake of succinic anhydride dissolves, add 28 ml 0.2 M sodium
borate (pH 8.0).
5. Mix briefly and immediately submerge slides in the solution, tap the tray to
dislodge any air bubbles that may be attached to the glass, and begin agitation
immediately, continuing for 15 minutes.
6. Pour off the blocking solution, rinse briefly with water, then transfer the rack
containing the microarrays to a 2 L beaker of containing room temperature water,
tap to remove air bubbles, and soak for 2 minutes.
7. Transfer the rack containing the microarrays and rinse briefly in 95% ethanol.
8. Dry the slides individually using compressed nitrogen. Stor e slides dry at room
temperature.
NOTES:
1. Salt crystals from the printing buffer make it possible to visualize the area on the
slide printed with DNA before blocking the slides. These crytals are dissolved
during blocking. If you are using hand hybridization under glass coverslips, you
should etch a line with a diamond-tipped pen on the back of the slide to mark the
area containing the DNA before blocking. Thus will make it easy to position a
coverslip before hybridization. If you are using an automated hybridization
system, the etching is not necessary.
2. Succinic anhydride has a very short half-life. This makes it important to have the
slides ready to immerse, the 0.2 M sodium borate ready to pipet, and to work
quickly. It is important to begin agitation immediately after adding the slides to the
blocking solution to avoid a phenomenon called "comet tails". Comet tails occur
when unbound DNA dissolves in the blocking buffer and binds to the glass
surface adjacent to where it was spotted, hence forming streaky tails from the
spots.
Return to list of protocols
Cy3 or Cy5-dUTP Direct Labeling Protocol Using Omniscript Reverse
Transcriptase
This protocol labels 2 ug of total RNA using Omniscript reverse transcriptase for use in
hybridizations on the Ventana Discovery System.
Materials required:
Omniscript RT™ Kit (Qiagen #205113)
Cy3-dUTP (Amersham #PA53032)
Cy5-dUTP (Amersham #PA55022)
2M HEPES (Ambion)
Montage PCR 96-well Cleanup (Millipore #LSKC09604)
10x dNTP mixture (made from 100 mM dNTP commercial stocks
2.5 N NaOH
Cy-3-labeled anchored oligo-dT(20)VN primer (Operon, custom synthesis)
Note: Use RNAse-free tubes and pipet tips
Program a PCR machine for 65oC 5min, 37oC 2 hr, and 37oC 15 minutes.
If a thermocycler is not available, heat one incubator to 65°C and a second
incubator to 37°C. Water baths, heat blocks, thermal cyclers, or hybridization
ovens (set to stationary) are all suitable for reverse transcription reactions as
long as they are capable of maintaining temperature within the suggested range
of ±1°C.
cDNA Labeling
1. Mix together in PCR RNase free tube (Ambion#12225):
 2 g total RNA
 Nuclease-free water (ex. Gibco #10977) to bring thevolume to 12.5 l
 1 l anchored oligo dT primer from a 500 ng/l stock concentration. The
final [oligo-dT] will be 0.5 M in 20 l volume.
Note: With other reverse transcriptases, we have used the same mass of
oligo dT(12-18) primer (Invitrogen #18418-012)
2. Heat to 65oC for 5 minutes.
3. Prepare the following reaction mixture and add to the above tube containing the
RNA and primer
10X RT buffer
2.0 l
10x dNTP mix (see below)
2.0 l
Cy3 or Cy5 dUTP (0.5 nmole)
0.5 l
40 U RNAse Inhibitor
1.0 l
4 U Omniscript RT
1.0 l
10x dNTP mix contains2.5 mM dATP, dCTP, dGTP and 1.5 mM TTP in water.
4. Incubate at 37oC for 2 hours.
5. Add 2 l 2.5 N NaOH and incubate at 37oC for 15 min.
6. Add 10ml 2M HEPES, vortex, and centrifuge briefly.
Note: At this point the samples may be stored at 4oC for several days.
Clean the labeled cDNA using Montage plates or Microcon spin columns.
Our preliminary experiments indicate that more genes were detected above
background on microarrays using the Montage system. The level of expression of
genes expressed in both systems was similar.
Montage PCR 96-well Cleanup Procedure
Details and troubleshooting may be found in the Millipore technical bulletin
#TN1600EN00 available on-line at
http://millipore.com/publications.nsf/docs/TN1600EN00.
1. Add 270 l water to each well to be used in the plate. You do not have to use an
entire plate. Wells not used should be left dry for future use. Once wells are used,
do not reuse them for other samples.
2. Add your sample (approximately 32 l) to the appropriate wells.
3. Place the Montage plate on a vacuum manifold and apply 10 inches mercury
vacuum until the wells are visually dry. Turn off the vacuum and allow the plate to
reach atmospheric pressure.
4. Add 25 l water per well. Cover the plate with an adhesive plate seal.
5. Shake the covered plate on a rotating shaking table for 10 minutes at room
temperature as fast as possible without allowing the samples to come into contact
with the seal (i.e. 1100 rpm on our IKA Shuttler MKS4 shaker).
6. Use pipets to transfer the resuspended nucleic acid to a microfuge tubes. The
volume should be between 20-25 l. If the volume is considerably larger, re-apply
the vacuum to the plate to further concentrate those particular sample(s).
Microcon Spin Column Cleanup Procedure.
1. Add 500 l TE (Ambion#9849) to a Microcon-100 column (Millipore, 42413).
2. Centrifuge for 3 minutes at 5000 x g and discard the flow-through.
3. Add 300 l TE to your labeled cDNA, and add pipet the solution to the prewashed
Microcon-100 column.
4. Centrifuge for 3 minutes, 5000 x g and discard the flow-through.
5. Wash the Microcon column with 400 l TE. Centrifuge for 3 minutes, 5000 x g and
discard the flow-through.
6. Wash the Microcon column again with 400 l TE. Centrifuge for 3 minutes, 5000 x
g and discard the flow-through.
7. Invert column into a fresh microfuge tube and centrifuge for 3 minutes, 5000 x g.
Carefully measure the volume of the probe. It should be <20 l. If it isn’t, put it
back in the Microcon and centrifuge for another minute at 5000 x g before
rechecking the volume.
To check the percent incorporation of cy3 and cy5 use the formula from the
following web site http://www.pangloss.com/seidel/Protocols/perc ent_inc.html
Return to list of protocols
Cy3 or Cy5-dUTP Direct Labeling Protocol Using EndoFree Reverse Transcriptase
This protocol labels 2 ug of total RNA using EndoFree reverse transcriptase for use in
hybridizations on the Ventana Discovery System.
Materials required:
EndoFree RT™ Kit (Ambion #1740)
Cy3-dUTP (Amersham #NEL578)
Cy5-dUTP (Amersham #NEL579)
500 mM EDTA (Ambion#9260G)
1 M Tris-HCl (pH 8) (Ambion#9855G)
Montage PCR 96-well Cleanup (Millipore #LSKC09604)
Note: Use RNAse-free tubes and pipet tips
Program a PCR machine for 70oC 5min, 42oC 65 m in, and 65oC 60 minutes.
If a thermocycler is not available, heat one incubator to 50°C and a second incubator
to 70°C Water baths, heat blocks, thermal cyclers, or hybridization ovens (set to
stationary) are all suitable for reverse transcription reactions as long as they are
capable of maintaining temperature within the suggested range ±1°C.
cDNA Labeling
1. Mix together in PCR RNase free tube (Ambion#12225):
 2 g total RNA
 Add nuclease-free water (ex. Gibco #10977) to bring the volume to 7 l
 1 l of anchored oligo dT primer supplied in the kit. The final [oligo-dT] will
be 0.5 M in 20 l volume.
Note: With other reverse transcriptases, we have used 1 ul of a 500 ng/ l
oligo dT12-18 primer) (Invitrogen Cat #18418-012)
2. Heat to 70oC for 10 minutes. Transfer quickly to 50oC and incubate for 10 min.
3. Prepare the following reaction mix and incubate at 50°C for 10 min
10X RT buffer
2.0 l
dCTP Solution
2.0 l
dGTP Solution
2.0 l
dATP Solution
2.0 l
dTTP Solution
0.6 l
Cy3 or Cy5 dUTP Solution 0.5 l
RNAse Inhibitor
2.0 l
EndoFreeRT
1.0 l
4. Pipette 12.1 µl of the reaction mixture (from step 3) into the RNA / primer mixture
(from step 2). Pipette solutions quickly to avoid cooling below ~48°C.
5. Incubate at 50oC for 2 hours.
6. Add 5 l 500mM EDTA (Ambion#9260G), vortex, and centrifuge quickly.
7. Add 10 l 1M NaOH, vortex, and centrifuge briefly. (EDTA and NaOH may be premixed immediatedly before addition).
8. Incubate at 65oC for 1 hour.
9. Cool to room temperature and add 25 l of 1M Tris-HCl (pH 8) (Ambion#9855G),
vortex, and briefly centrifuge the samples.
Note: At this point the samples may be stored at 4oC for several days.
Clean the labeled cDNA using Montage plates or Microcon spin columns.
Our preliminary experiments indicate that more genes were expressed above
background on microarrays using the Montage system. The level of expression of
genes expressed in both systems was similar.
Montage PCR 96-well Cleanup Procedure
Details and troubleshooting may be found in the Millipore technical bulletin
#TN1600EN00 available on-line at
http://millipore.com/publications.nsf/docs/TN1600EN00.
1. Add 240 l water to each well to be used in the plate. You do not have to use an
entire plate. Wells not used should be left dry for future use. Once wells are
used, do not reuse them for other samples.
2. Add your sample (approximately 60 l) to the appropriate wells.
3. Place the Montage plate on a vacuum manifold and apply 10 inches mercury
vacuum until the wells are visually dry. Turn off the vacuum and allow the plate to
reach atmospheric pressure.
4. Add 25 l water per well. Cover the plate with an adhesive plate seal.
5. Shake the covered plate on a rotating shaking table for 10 minutes at room
temperature as fast as possible without allowing the samples to come into
contact with the seal (i.e. 1100 rpm on our IKA Shuttler MKS4 shaker).
6. Use pipets to transfer the resuspended nucleic acid to a microfuge tubes. The
volume should be between 20-25 l. If the volume is considerably larger, re-apply
the vacuum to the plate to further concentrate those particular sample(s).
Microcon Spin Column Cleanup Procedure.
1. Add 500 l TE (Ambion#9849) to a Microcon-100 column (Millipore, 42413).
2. Centrifuge for 3 minutes at 5000 xg and discard the flow-through.
3. Add 300 l TE to your labeled cDNA, and add pipet the solution to the
prewashed Microcon-100 column.
4. Centrifuge for 3 minutes, 5000 x g and discard the flow-through.
5. Wash the Microcon column with 400 l TE. Centrifuge for 3 minutes, 5000 x g
and discard the flow-through.
6. Wash the Microcon column again with 400 l TE. Centrifuge for 3 minutes, 5000
x g and discard the flow-through.
7. Invert column into a fresh microfuge tube and centrifuge for 3 minutes, 5000 x g.
Carefully measure the volume of the probe. It should be <20 l. If it isn’t, put it
back in the Microcon and centrifuge for another minute at 5000 x g before
rechecking the volume.
To check the percent incorporation of cy3 and cy5 use the formula from the
following web site http://www.pangloss.com/seidel/Protocols/percent_inc.html
Return to list of protocols
OMRF Modified CyScribe Post cDNA Labeling Protocol
1.
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Resuspend a tube of the AA-dUTP from the kit in 30 ml nuclease free water. This
is good for 30 days from the time resuspended and can be used for other
reactions.
Add the following components to a 0.2 ml PCR tube:
10-20 g Total RNA
X l
Anchored oligo (dT)
3 l
Water
Y l
11 l
Mix gently and spin briefly.
Incubate in PCR cycler 70 C for 5 minutes.
Cool reactions to 20 C for 10 minutes to allow primers to anneal.
Briefly spin tubes and place on ice.
Add the following components (make premix but add enzyme just before use):
1X
5X CyScript buffer
4 l
0.1 M DTT
2 l
Nucleotide mix
1 l
AA-dUTP
1 l
RNaseOUT
1 l
CyScript RT
1 l
10  l
Mix gently and spin briefly.
Incubate reactions at 42 C for 1.5 hours.
Add 2 l 2.5 M NaOH to each reaction.
Mix gently and spin briefly.
Incubate reactions at 37 C for 15 minutes.
Add 10 l 2 M HEPES free acid to each reaction.
Mix by vortexing, spin briefly, then transfer contents to 0.5 ml tube.
Add the following components to each reaction:
Glycogen (20 mg/ml)
1 l
3 M Sodium Acetate
3 l
95% Ethanol
100 l
Chill reactions 15 minutes on dry ice.
Centrifuge reactions 15 minutes at 4 C, wash pellets 1X in cold 70% Ethanol.
Centrifuge another 15 minutes at 4 C, decant and air dry pellet.
Resuspend the pellet in 15 l water.
Resuspend 1 aliquot of Cy3 or Cy5 dye in 15 l 0.1 M Sodium bicarbonate pH 9.0.
Add Cy dye to cDNA and incubate 1 hour in the dark at room temperature. Use
Cy5 for control sample and Cy3 for experimental sample.
22. Clean up each reaction separately with the Rapid PCR Purification System
(Marligen Bioscience Inc, catalog #11458-015):
Preheat aliquot of water to 65-70 C.
Add 400 l H1 binding solution, mix well.
Load sample into cartridge in a 2 ml tube.
Centrifuge for 1 minute @ max speed, discard flow through.
Add 700 l wash buffer to cartridge.
Centrifuge 1 minute @ max speed, discard flow through.
Centrifuge again for 1 minute @ max speed to remove residual buffer.
Place cartridge into a fresh 1.5 ml tube.
Add 50 l warm water to the center of the cartridge, incubate 5 minutes.
Centrifuge 2 minutes @ max speed. Discard cartridge.
23. Combine Cy3- and Cy5-labeled cDNAs that will be used on the same slide.
24. Read OD260, OD550, and OD649 on the combined 100 l of cDNAs (no dilution).
25. Recover cDNA from the cuvette and add the following components:
Yeast tRNA (4 mg/ml)
1 l
Poly d(A)40-60 (8 mg/ml)
1 l
Human or mouse COT-1 (1 mg/ml)
10 l
3 M Sodium Acetate
11 l
95% Ethanol
260 l
26. Chill 15 minutes on dry ice.
27. Centrifuge for 15 minutes at 4 C, decant and air dry pellet.
28. Resuspend the pellet in appropriate amount of GlassHyb hybridization solution
(Clontech) for the array size -- e.g., 24 x 60 mm lifterslips # 24X60I require ~80 l,
while lifterslips #24X60IS require ~ 65 l.
Note: there is variability in these coverslips and thus variability in volume.
29. Vortex well. Pellet may not dissolve until heated.
30. Heat 2 minutes at ~90oC (solution will become white), vortex, and then incubate for
10 minutes at 65o C (solution will t urn clear again).
31. While the hybridization solution is at 65o C, prepare the slide, lifterslip, and hyb
chamber. Place slide in chamber and align lifterslip to cover array. Pipet 20 l
water into the 2 circular reservoirs within the hyb chamber.
32. After the 65o C incubation, transfer the hyb solution to the slide. Seal hyb chamber
and place in water bath at 65o C. Incubate overnight (~16-18 hr).
Washes:
33. The next day, follow Clontech’s GlassHyb wash protocol in 50 ml conical tubes.
34. Transfer slide & lifterslip to 50 ml tube containing 33 ml undiluted Clontech
GlassHyb wash buffer (2X SSC/ 1% Tween 20) at room temperature. After a brief
soak the lifterslip should come loose. Grab the slide, lift it up, and place in front of
the lifterslip. This will allow you to agitate the tube and not have the lifterslip rub the
microarray spots since the lifterslip should now be on the opposite side of the slide
as the array.
35. Lay tube horizontal in rack on orbital shaker and shake 10 minutes.
36. Wash 2 x 10 min in 33 ml of the following buffer:
3 ml GlassHyb diluted into 30 ml 1X SSC; room temp, shaking.
37.
38.
39.
40.
Wash for 10 min in 33 ml 0.1X SSC, room temp, shaking.
Rinse 2 x in 33 ml water briefly, shaking (~2 min each rinse).
Spin dry for 5 minutes at ~500xG.
Transfer to slide carrier.
Store slides in the dark at room temperature until scanned.
Return to list of protocols
OMRF Automated Microarray Hybridization Protocol
The OMRF Microarray Facility uses the Ventana DiscoveryTM system for hybridization.
This machine offers advantages of automated liquid-handling, individually temperature
controlled pads for each microarray, and continuous mixing of hybridization solution
under an oil-based cover slip. Hybridization steps include preparation of cDNA for
hybridization after labeling, placement of the microarray and cDNA on the Discovery
machine, automated steps carried out by the Discovery machine, and final manual
washing steps of the microarrays prior to scanning.
Preparation of Hybridization Blocking Master Mix
Materials Needed
Poly d(A)40-60 (Amersham # 27-7988-01)
Yeast tRNA (10 mg/ml) (Ambion # 7119)
Cot-1 (1 mg/ml) (Invitrogen # 15279-011 [Human] or 18440-016 [Mouse])
50X Denhardts Solution (Sigma # D-2532)
Deionized Formamide (Sigma # F-9037)
ChipHybeTM buffer (Ventana Medical # 760-103)
20X SSC (Ambion # 9763)
Protocol
1. Dilute Poly d(A) to 100 mg/ml in water
2. Prepare Blocking Master Mix (for 100 samples)
Poly d(A) (100 mg/ml)
8 µl
Yeast tRNA (10 mg/ml)
40 µl
Cot-1 (1 mg/ml)
1.0 ml
Note: The hybridization blocking master mix may be prepared ahead of time and
stored in aliquots at -20C for future use.
Default Ventana Discovery Hybridization and Washing Protocol
Manual Steps
Load barcoded microarray slides on Ventana heat pads.
Load the ChipPrepTM1, ChipPrepTM2, and ChipCleanTM tubes on the Ventana
reagent carousel.
Press start.
Automated Steps
Wet slide with SSPE-based buffered solution
Apply ChipPrepTM1 and coverslip, 70C for 10 min
Rinse slides
Apply ChipPrepTM2 and coverslip, incubate 30 min
Rinse slides
Apply coverslip
Manual Step
Add all of the cDNA (~24 µl) to 176 µl of master hybridization solution in a
microfuge tube. Master hybridization solution is prepared by mixing:
H2O
37.5 µl
50x Denhardts
8.0 µl
Deionized Formamide 20.0 µl
ChipHybe
100.0 µl
Blocking Master Mix
10.5 µl
Total
176 µl
Heat to 95C for 5 min
Centrifuge at 10,000 rpm for 5 min at room temperature
Transfer tubes to 58C until ready to apply to the microarray. Apply the solution
under the oil-based liquid cover slip.
Automated Steps
Warm slides to 37C for 6 min
Warm slides to 42C for 2 min (denaturation)
Warm slides to 58C for 9 hr (hybridization)
Stringency Wash 1, 37C for 2 min
Rinse slides
Apply ChipCleanTM, 37C for 2 min
Manual Steps
Remove microarrays from the slide holder and wipe oil from the back with a
kimwipe.
Place slide in slide holder rack immersed in Ventana Reaction Buffer
Plunge slides up and down 25 times in:
- Ventana Reaction Buffer
- Ventana Reaction Buffer
- 1X SSC
Plunge slides up and down 2 times in 0.1X SSC
Remove one slide at a time from 0.1x SSC and blow dry with compressed
nitrogen gas.
Return to list of protocols
cDNA Synthesis for Quantitative Real-Time PCR (QRT-PCR) using Omniscript
Reverse Transcriptase
The cDNA synthesis in this protocol is similar to the Direct Cy-Labeling Protocol Using
Omniscript Reverse Transcriptase that is used for our microarrays. Our QRT-PCR
protocol may be accessed here
Materials required:
 OmniScript RT™ Kit (Qiagen #205113)
 10X dNTP Mix (2.5 mM each in TE, pH 8.0; 100 mM stocks from Invitrogen)
 RNAsin RNAse Inhibitor (Promega, 40U/ml)
 Anchored Oligo dT Primer dT20VN (Operon, custom synthesis where V = G, C or
A; N = G, C, A or T)
 Nuclease-Free Water
 2.5 N NaOH
 2M HEPES (Ambion, or made with nuclease-free water)
 Montage PCR 96-well Cleanup (Millipore #LSKC09604)
NOTE: Use only RNAse-free tubes and pipets
Program the PCR machine
 65°C, 5 min
 37°C, 2 hr
 37°C, 15 min
If a thermocycler is not available heat one incubator to 65°C and a second incubator
to 37°C. Water baths, heat blocks, thermal cyclers, or hybridization ovens (set to
stationary) are all suitable for reverse transcription reactions as long as they are
capable of maintaining temperature within the suggested range ±1°C.
cDNA Synthesis
1. Mix together in PCR RNase free tube (Ambion#12225):
 1 g total RNA
 Add nuclease-free water (ex. Gibco #10977) to bring the volume to 13 l
 1 l of anchored oligo dT primer (500 ng/ml stock conc.). The final [oligo-dT]
will be 0.5 mM in 20 l volume.
2. Heat to 65ºC for 5 minutes.
3. Prepare the following reaction mix and add to the above tube containing the RNA
and primer:
 2 l 10X RT buffer
 2 l 10X dNTPs
 1 l (40 U) RNAse Inhibitor
 1 l (4 U) OmniScript RT
4. Incubate at 37ºC for 2 hours.
5. Add 2 ml 2.5 N NaOH and incubate at 37ºC for 15 min.
6. Add 10 ml 2M HEPES, vortex, and centrifuge briefly.
Note: At this point the samples may be stored frozen for several days.
7. Clean up the cDNA using Montage plates.
Montage PCR 96-well Cleanup Procedure
Details and troubleshooting suggestions may be found online in the Millipore technical
bulletin #TN1600EN00.
1. Add 270 l water to each well to be used in the plate. You do not have to use an
entire plate. Wells not used should be left dry for future use. Once wells are
used, do not reuse them for other samples.
2. Add your sample (approximately 32 l) to the appropriate wells.
3. Place the Montage plate on a vacuum manifold and apply 10 inches mercury
vacuum until the wells are visually dry. Turn off the vacuum and allow the plate to
reach atmospheric pressure.
4. Add 25 l water per well. Cover the plate with an adhesive plate seal.
5. Shake the covered plate on a rotating shaking table for 10 minutes at room
temperature as fast as possible without allowing the samples to come into
contact with the seal (i.e. 1100 rpm on our IKA Shuttler MKS4 shaker).
6. Use pipets to transfer the resuspend nucleic acid to a microfuge tubes. The
volume should be between 20-25 l. If the volume is considerably larger, re-apply
the vacuum to the plate to further concentrate those particular sample(s).
7. Dilute cDNA 1:20 in water. Use 2 l for each PCR reaction.
Note: We have used Montage plates and Microcon spin columns to clean up labeled
cDNA for microarrays. Our preliminary experiments suggested that more genes were
detected above background on microarrays using the Montage system. The signal
intensities of genes were similar.
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SYBR® Green I Quantitative Real-Time PCR (QRT-PCR) Protocol
Real-time PCR amplification is done on an ABI®PRISM 7700 using the SYBR® Green I
dye assay chemistry. All samples are run in triplicate with the appropriate RT-PCR
controls run in singlet (no reverse transcriptase, no template, etc.). Parallel reactions
are run from the same cDNA sample for the gene of interest and a control gene for
normalization. Following the RT-PCR, dissociation curve analysis is performed to
confirm amplification of a single gene product.
PCR Reaction (15 l volume per gene):
Prepare two master mixes: one for the gene of interest and one for the control gene.
1X Master Mix



7.5 l SYBR® Green I PCR Master Mix [Applied Biosystems (ABI) # 4309155]
5.2 l Water
0.3 l Mixture of 5' primer + 3' primers (50 pmol each/ ml)
Add 13 l of master mix per well to a 96-well optical reaction plate (ABI # 4306737). Add
2.0 l of cDNA template to each well. Cover the wells with optical caps (ABI #4323032).
Mix and centrifuge the plate briefly (a few seconds at 1000 rpm).
Cycling Conditions:
 1 cycle
95°C for 10 minutes
 40 cycles
95°C for 15 seconds
60°C for 1 minute
 1 Cycle (Dissociation Curve)
95°C for 15 sec
60°C for 20 sec
95°C for 15 sec [ramp 20 min]
QRT-PCR Data Analysis
Using the QRT-PCR data, an average Ct value is calculated from triplicate
reactions. Averaged Ct values are then normalized (to adjust for different amounts of
cDNA in each reaction) to averaged Ct values for a control gene. Adjusted average Ct
values are used to calculate the average of the biological replicates, and then the Ct is
calculated between those groups. This number is anti-logged to calculate fold change.
(In general, if the Ct values are within 0.5 units of each other, there is little need to
adjust the Ct values. Relative expression levels of the same gene between 2 samples
are determined according to Ct values. A Ct value < 1 is considered no change.)
QRT-PCR Primer Design Notes
Primers should be localized around the microarray oligonucleotide and follow these
general primer design rules:
 no more than 2 G's or C's within the last 5 bases





no more than 3 consecutive G's within the primer
the 3' end of each primer must not end in a G or a C
the optimal Tm of the primer is 58 to 60°C
the optimal primer length is 20 base pairs (range 17-25 bp's)
all primers are designed to generate 70-110 bp products.
We use the free Netprimer Software to check for primer dimers, hairpins, and cross
dimers. This software generates a rating for each primer from 0 to 100%. Most of our
primers rate 90% or above; however, we do have some primers that work well and
generated a rating of 85%. We have found this software to work very well for predicting
good QRT primers.
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Validation of Microarray Results Using Quantitative Real-Time PCR (QRT PCR)
with EndoFree RT
cDNA Synthesis
RNA used for quantitative real-time PCR should be identical to the RNA used on the
microarray. The following protocol is slightly modified from the Direct Cy-Labeling
Protocol Using EndoFree Reverse Transcriptase listed on our microarray website.
Modifications include the addition of 0.1ng of plant mRNA (Stratagene) for QRT PCR
normalization, the use of equimolar amounts of dNTPs, and the use of H 20 instead of a
Cy-labeled dNTP.
1. Program the PCR machine
 70°C 5 min
 50°C 130 min
 65°C 60 min
Notes: Use RNase free tubes and tips. If a thermo cycler is not available heat
one incubator to 50°C and a second incubator to 70°C. Water baths, heat blocks,
thermal cyclers, or hybridization ovens (set to stationary) are all suitable for
reverse transcription reactions as long as they are capable of maintaining
temperature within the suggested range ±1°C.
2. Prepare RNA:RTPrimer Mix
 Place 2 µg of test RNA and 0.1 ng of plant mRNA in a 0.5 ml RNase-free
microfuge tube. If necessary, add Nuclease-free water to the RNA to bring
the volume to 7 µl. The volume of the RNA may not exceed 7 µl per
reaction. Add 1 µl of the anchored oligo d(T) primer supplied with the kit
(final conc. 0.5 µm).
 Incubate at 70°C for 10 min
 Quickly move mixture to 50°C and incubate 10 min
3. Prepare reaction mix and incubate at 50°C for 10 min
 10X RT Buffer
2 µl
 dCTP Solution
2 µl
 dGTP Solution
2 µl
 dATP Solution
2 µl
 dTTP Solution
2 µl
 RNase Inhibitor
2 µl
 Endo-Free RT™
1 µl
4. Add reaction mix to RNA:RT primer mix
 Pipette the reaction mix (from step 3) into the RNA:RT primer mix (from
step 2).
 Pipette solutions quickly to avoid cooling below ~48°C.
5. Incubate at 50°C for 2 hours
6. Add 5 µl 500 mM EDTA & 10 µl 1 M NaOH
7. Incubate at 65°C for 1 hour
8. Cool to room temperature and add 25 µl of 1 M Tris-HCl, pH 8.0, vortex and
centrifuge briefly
Montage PCR 96-well Cleanup Procedure
Details and troubleshooting may be found in the Millipore technical bulletin
#TN1600EN00 available on-line at http://millipore.com/publications.nsf/docs/
TN1600EN00. Details and troubleshooting may be found in the Millipore technical
bulletin #TN1600EN00.
1. Add 240 µl water to each well to be used in the plate. You do not have to use an
entire plate. Wells not used should be left dry for future use. Once wells are
used, do not reuse them for other samples.
2. Add your sample (approximately 60 µl) to the appropriate wells.
3. Place the Montage plate on a vacuum manifold and apply 10 inches mercury
vacuum until the wells are visually dry. Turn off the vacuum and allow the plate to
reach atmospheric pressure.
4. Add 25 µl water per well. Cover the plate with an adhesive plate seal.
5. Shake the covered plate on a rotating shaking table for 10 minutes at room
temperature as fast as possible without allowing the samples to come into
contact with the seal (i.e. 1100 rpm on our IKA Shuttler MKS4 shaker).
6. Use pipets to transfer the resuspended nucleic acid to a microfuge tubes. The
volume should be between 20-25 µl. If the volume is considerably larger, re-apply
the vacuum to the plate to further concentrate those particular sample(s).
QRT PCR Primer Design
All primers are localized around the microarray oligonucleotide and follow these primer
design rules:
 no more than 2 G's or C's within the last 5 bases
 no more than 3 consecutive G's within the primer
 the 3' end of eac h primer must not end in a G or a C
 the optimal Tm of the primer is 58 to 60°C
 the optimal primer length is 20 base pairs (range 17-25 bp's)
 all primers are designed to generate 70-110 bp products.
We use the free Netprimer Software to check for primer dimers, hairpins, and cross
dimers. This software generates a rating for each primer from 0 to 100%. Most of our
primers rate 90% or above; however, we do have some primers that work well and
generated a rating of 85%.. We have found this software to work very well for predicting
good QRT primers.
QRT PCR
Real-time PCR amplification is done on an ABI®PRISM 7700 using the
SYBR®Green I dye assay chemistry. All samples are run in triplicate with the
appropriate single RT PCR controls (no reverse transcriptase, no template, no oligo-dT,
etc.). Following the RT PCR, dissociation curve analysis is performed to confirm the
desired single gene product.
PCR Reaction (15 ml total volume):
 7.5 µl SYBR®Green PCR Mix (ABI # 4309155)
 4.9 µl Water
 0.3 µl 5' primer [50 pmol/ul]
 0.3 µl 3' primer [50 pmol/ul ]
 2.0 µl Template [1 ng]
We use more than the recommended (5 pmol/µl) amount of primer. This is overkill but it
is what we have found works well for us.
Cycling Conditions:
 1 cycle
95°C for 10 minutes
 40 cycles
95°C for 15 seconds
60°C for 1 minute
 1 Cycle (Dissociation Curve)
95°C for 15 sec
60°C for 20 sec
95°C for 15 sec
QRT PCR Data Analysis
From the QRT PCR data, an average Ct value is calculated from the triplicate
reactions. Averaged Ct values are then normalized (to adjust for different amounts of
cDNA in each reaction) to a plant control gene, RCA, RCP1, or NAC1. If the Ct values
are within 0.5 units of each other, there is no need to adjust the Ct values. Relative
expression levels of the same gene between 2 samples are determined according to
Ct values. A Ct value < 1 is considered no change.
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