NimbleGen 1-8
Gene Expression Analysis
Synthesis of Double-Stranded cDNA
Outline:
This protocol describes the process for hybridization of gene expression samples to
NimbleGen DNA microarrays. The first step of this process is creating cDNA from
Eukaryotic Total RNA or Poly A+ RNA and cDNA from Prokaryotic Total RNA using
Invitrogen’s SuperScriptTM Double-Stranded cDNA Synthesis Kit. The second step of the
process labels the cDNA and hybridizes to a NimbleGen expression array using the
MAUI Hybridization System. The array is stringently washed, dried, and scanned.
Protocol Information & Safety:
 Wear gloves to avoid sample contamination of degradation.
 RNA should be kept on ice at all times.
 Phenol:chloroform:isoamyl alcohol is dangerous and should be handled with caution.
Wear the correct personal protective equipment when handling this chemical.
Required Apparatus & Labware:
 Thermocycler
 Microcentrifuge with 14,000 x g capability
 Gel apparatus
 Vortexer
 Spectrophotometer
 Vacuum Centrifuge
Step 1. Spectrophotometric QC of RNA:
Prior to creating cDNA, it is necessary to verify that the RNA samples are of sufficient
purity to proceed with cDNA synthesis.
1. Blank the spectrophotomer.
2. Take readings for each RNA sample.
3. Verify that all samples meet the following requirements
 A260/A280 greater than or equal to 1.7
 A260/A230 greater than or equal to 1.5
Step 2. Bioanalyzer QC of RNA:
It is also necessary to verify that the RNA samples are of sufficient molecular weight
prior to creating cDNA.
1. Transfer 25 – 500ng Total RNA of 25-250ng Poly A+ RNA to a sterile
microcentrifuge tube. Store the remainder of your sample on ice or at -80oC.
2. If using a denaturing agarose gel, a possible source for reagents is Ambion’s
NorthernMax reagents for northern blotting. The first section of this kit includes
reagents and instructions for running denaturing agarose gels.
3. Review the Bioanalyzer tracer or denaturing gel or gel images in comparison to
those displayed below. Degraded samples appear as significantly lower intensity
tracer with the main peak area shifted to the left and typically exhibit much more
noise in the trace.
NimbleGen 2-8
Samples exhibiting degradation should no be carried through labeling and
hybridization, because there is an unacceptable risk of poor results.
Step 3. First Strand cDNA Synthesis:
Use the Invitrogen SuperscriptTM Double-Stranded cDNA Synthesis Kit or another
preferred kit to synthesize double-stranded cDNA.
For Invitrogen Kit
1. Thaw and maintain the following components on ice. Combine components in an
RNase-free 1.5 ml microcentrifuge tube according to the tables below.
Eukaryotic Source of RNA
Volume
10 ug Total RNA
oligo dT Primer
1ul
DEPC-Water
To volume
Total
11ul
2.
3.
4.
5.
6.
7.
8.
Prokaryotic Source of RNA
Volume
10 ug Total RNA
Random Primer
1ul
DEPC-Water
To volume
Total
11ul
Heat sample(s) to 70˚C for 10 minutes. Briefly spin tubes in a microcentrifuge,
and place the reaction(s) in an ice water slurry.
Add the following to each reaction (a master mix may be used):
Volume
Reaction volume from Step 3.2
11ul
5X First Strand Buffer
4ul
0.1M DTT
2ul
20mM dNTP mix
1ul
Cumulative Total
18ul
Mix gently (avoid vortexing). Briefly spin the tube(s) in a microcentrifuge.
Place the sample(s) in a thermocycler set at 42oC for 2 minutes.
Add 2 ul of SuperScript II and mix gently (avoid vortexing).
Incubate the sample(s) at 42oC for 60 minutes.
Briefly spin the tube(s) in a microcentrifuge. Place the sample(s) on ice until the
second strand synthesis. Sample(s) may be stored at -20oC overnight.
Good stop point if needed
Step 4. Second Strand cDNA Synthesis:
1. Add the following components to the first strand reaction(s) in the indicated order
(a master mix may be used). Keep tube(s) on ice or in a PCR tube chiller rack.
Volume
Reaction from Step 3.8
20ul
DEPC-Water
91ul
NimbleGen 3-8
5X Second Strand Buffer
30ul
10mM dNTP mix
3ul
10U/ul DNA Ligase
1ul
10U/ul DNA Polymerase I
4ul
2U/ul RNase
1ul
Cumulative Total
150ul
2. Mix gently (avoid vortexing). Briefly spin the tube(s) in a microcentrifuge.
Incubate at 16oC for two hours.
3. Add 2ul of 5U/ul T4 DNA polymerase to each reaction. Incubate at 16oC for an
additional 5 minutes. Do not allow the reaction temperature to exceed 16oC
during this step.
4. Place the sample(s) on ice or PCR tube chiller rack, and add 10ul of 0.5M EDTA.
Samples may be stored at -20oC overnight.
Good stop point if needed
Step 5. RNase A Clean-up:
1. Ad 1 ul of 10 mg/ml RNase A solution to the tubes.
2. Mix gently (avoid vortexing). Briefly spin the tube(s) in a microcentrifuge.
3. Incubate sample(s) at 37oC for 10 minutes.
4. During incubation, centrifuge Phase Lock tube(s) for two minutes at maximum
speed. Label the phase lot tube(s) and two sets of 1.5 mL centrifuge tubes with
sample names.
5. Add 63 ul of Phenol: Chloroform: Isoamyl Alcohol to one set of 1.5mL centrifuge
tubes.
6. Transfer the sample(s) to the tubes containing Phenol: Chloroform: Isoamyl
Alcohol.
7. Mix. Transfer samples with the Phenol: Chloroform: Isoamyl Alcohol to Phase
lock Tubes.
8. Centrifuge at maximum speed for five minutes.
9. Transfer the upper, aqueous layer to a clean 1.5 mL tube.
Step 6. cDNA Precipitation:
1. Add 6 ul of 7.5 M Ammonium Acetate to the samples. Mix by repeated inversion.
Briefly spin the tube(s) in a microcentrifuge.
2. Add 7 ul of 5mg/ml Glycogen to the samples. Mix by repeated inversion. Briefly
spin the tube(s) in microcentrifuge.
3. Add 326 ul of ice-cold absolute Ethanol to the samples. Mix by repeated inversion.
4. Centrifuge at maximum speed for 20 minutes.
5. Remove supernatant. Take care not to disturb the pellet.
6. Add 500 ul of ice-cold 80% Ethanol (v/v). Mix by repeated inversion.
7. Centrifuge tubes at maximum speed for five minutes.
8. Remove the supernatant. Take care not to disturb the pellet.
9. Repeat steps 6,7,and 8.
10. Dry Pellet in SpeedVac.
11. Rehydrate samples with 20 ul of DI water (VWR)
NimbleGen 4-8
Step 7. Spectrophotometric QC of cDNA:
1. Blank the spectrophotometer.
2. Measure each cDNA sample.
3. Verify that all samples meet the following requirements:
 Concentration ≥ to 100 ng/ ul
 A260/A280 ≥1.7
 A260/A230≥1.5
Step 8. Bioanalyzer/Gel QC of cDNA:
1. Transfer 100-250ng cDNA to a sterile microcentrifuge tube. Store the remainder
of your samples on ice or at -20oC.
2. Analyze the samples using the Agilent Bioanalyzer or Agarose Gel.
3. Compare the Bioanalyzer traces or gel images, to those displayed below. Verify
that all the samples meet the following requirements for acceptance:
 Median size ≥ 400 bp when compared to DNA ladder.
 Look similar to the examples of good cDNA sample traces or gels listed
below.
Samples exhibiting degradation should not be carried through labeling and
hybridization, because there is an unacceptable risk of poor results.
Step 9. Labeling of cDNA:
1. Prepare the following three solutions:
50 Dnpt Mix
Volume Aliquot 50x dNTP mix into single-use
VWR deionized water
250 ul amounts. Avoid freeze-thaw cycles,
10X TE Buffer
50 ul which can result in diminished yields.
100 mM dATP
50 ul
100 mM dGTP
50 ul
100 mM dTTP
50 ul
100 mM dCTP
50 ul
Total
500ul
Random 9mer Buffer
VWR deionized water
1M Tris-HCL, Ph7.4
1M MgCl2
Β-Mercaptoethanol
Total
Volume Make fresh each time primers are
8.6ml diluted.
1.25ml
125ul
17.5ul
10ml
10X TE Buffer
Volume Mix and Store at room temperature.
1M Tris-HCL, PH7.4
1.5 ml
0.5M EDTA
300ul
VWR deionized water
13.2 ml
Total
15ml
2. Dilute Cy3 dye-labeled 9mers to 1 O.D./42 ul Random 9mer Buffer. Aliquot 40 ul
in 0.2 ml thin-wa;;ed PCR tubes and store at -20oC.
3. Assemble the following components in separate 0.2ml thin-walled PCR tubes.
NimbleGen 5-8
Volume
1 ug cDNA
Cy3-9mer Primers
40ul
VWR Water
To Volume
Total
80 ul
o
4. Heat denature samples in thermocycler at 98 C for 10 min. Quick Chill the
samples in an ice water bath. Add the following ( a master mix may be used):
Volume
Reaction Volume from step 2.3
80 ul
50X nNTP mix
10 ul
VWR deionized water
8 ul
Klenow (50 U/ ul)
2 ul
Cumulative Total
100 ul
5. Mix well by pipetting up and down ten times, do not vortex.
6. Centrifuge at low RPM to collect the contents to the bottom of the tube.
7. Incubate at 37oC for 2 hours in thermocycler. Protect from light.
8. Stop the reaction by addition of EDTA:
Volume
Reaction Volume from step 2.4
100 ul
0.5M EDTA
10 ul
Cumulative Total
110 ul
9. Transfer the reaction to a 1.5 ml tube. Precipitate by adding NaCl indicted and
vortex briefly. Then add isopropanol to each tube as indicated.
Volume
Reaction volume from step 2.7
110 ul
5M NaCl
11.5 ul
Isopropanol
110 ul
Cumulative Total
231.5 ul
10. Vortex. Incubate at room temperature for ten minutes. Protect from light.
11. Centrifuge at maximum speed for ten minutes. Remove supernatant with pipette.
12. Rinse pellet with 500 ul 80% ice-cold ethanol. Dislodge pellet from tube wall.
13. Centrifuge at maximum speed for 2 minutes. Remove supernatant with pipette.
14. Vacuum centrifuge on low heat for five minutes until dry. Protect from light.
15. Store labeled samples at -20oC. Protect from light.
16. Briefly spindown tubes if stored at - 20oC. Re-hydrate dried pellets in 25 ul VWR
deionized water.
17. Vortex for 30 seconds. Briefly spin the tube(s) in a microcentrifuge. Continue to
vortex or let sit in the dark at room temperature until the pellet is completely
rehydrated, then vortex again and quickly spin.
18. Determine the concentration of each sample. The concentration can be determined
using the following formula.
Concentration (ul/ml)=A260 X 50 X Dilution Factor
Step 10. Hybridization of Cy-labeled DNA:
NimbleGen 6-8
IMPORTANT!- Protect tube from light during handling to prevent photo bleaching of the
Cy dyes.
1. Set MAUI Hybridization System to 42oC and allow the temperature to stabilize.
2. Based on the concentration determined in step 9.18 and the table below, calculate
the amount of sample required. Place sample into 1.5ml tubes. \
Sample Requirements
Amount
Eukaryotic Sample
13ug
Prokaryotic Sample
1.5ug
3. If the volume is larger than 11.2 ul, dry the contents in a vacuum centrifuge on
low heat. Resuspend the sample in 11.2ul VWR water and vortex to dissolve.
Briefly spin the tube(s) in a microcentrifuge.
4. If the volume is smaller than 11.2ul, bring the volume up to 11.2ul with water
(VWR). Briefly spin the tube(s) in a microcentrifuge.
5. Using the NimbleGen Hybridization Kit, add the following to the suspended
sample:
Hybridization Solution
Volume
Cy labeled sample
11.2 ul
2X Hybridization Buffer
19.5 ul
Hybridization Component A
7.8 ul
Cy3 CPK6 48mer Oligo, 50nM
0.5 ul
Total
39 ul
IMPORANT!- CPK6 48mer Oligos are included in the hybridization as controls that
hybridize to alignment features on NimbleGen arrays. They are required for proper
extraction of the array data from the scanned image.
6. Mix. Briefly spin the tube(s) in a microcentrifuge. Place at 95oC for 5 minutes.
7. Immediately transfer the tube to the MAUI 42oC sample block and hold at this
temperature until you are ready for sample loading.
8. Place the MAUI Mixer SL Hybridization Chamber on the array using the
provided assembly/disassembly jig and carefully follow MAUI setup instructions.
Use the braying tool to remove all air bubbles from the adhesive gasket around
the outside of the hybridization chamber.
9. Put the array and Hybridization Chamber on the MAUI and allow 30 seconds for
the chips to come up to temperature.
IMPORANT!- Hybridization chambers have a finite shelf life. Check the expiration date.
Use the chambers within 30 minutes of opening the package.
10. Load the sample using the pipette supplied with the MAUI Station; follow the
manufacturer’s instructions. During loading, a small amount of (3-7 ul) of the
sample may flow out of the outlet port.
11. Confirm that there are no bubbles in the chamber. If present, very gently, message
any bubbles to either of the ends, away from the center of the array. Avoid
applying too much pressure because this will force liquid out of the ports.
12. Place the loaded array into one of the four MAUI bays and let equilibrate for 30
seconds. Wipe off any sample leakage at the ports, and adhere the MAUI stickers
to both ports.
13. Close the bay clamp and select mix mode B. Hold down the mix button to start
mixing. Confirm that the mixing is in progress before closing the cover.
NimbleGen 7-8
14. Hybridize the sample overnight (16-20 hours).
Step 11. Washing of Arrays:
1. Prior to removing the array from the MAUI Hybridization station, prepare the
following solutions. You will need two 250 ml dishes of Wash I, and one for each
for Wash II and Wash III. One dish for Wash I should be shallow and wide
enough to accommodate the array and mixer loaded in the MAUI
assembly/disassembly jig. The lid from a 1000 ul pipette tip box works well.
Also, the buffer in the first Wash I dish should be heated to 42oC to help soften
the adhesive on the hybridization chamber; this will help prevent braking the
array. Place the remaining three wash solutions in 300ml Tissue-Tek slide
staining dishes.
Wash I X 2
Wash II
Wash III
VWR water
225ml
225ml
225ml
10X Wash Buffer I,II, or III
25ml
25ml
25ml
1 M DTT
25 ul
25 ul
25 ul
Total
250ml
250ml
250ml
2. Remove the chip from MAUI Hybridization Station and load it back to the MAUI
assembly/disassembly jig. Immerse in the shallow 250ml Wash I at 42oC. While
the chip is submerged, carefully, peel off the mixer hybridization chamber.
IMPORANT!-Peel the hybridization chamber off very slowly to prevent the slide from
cracking. Do not let the surface of the slide dry out at any point during washing.
3. Transfer the slide into a slide rack in the second dish of Wash I. Incubate 2
minutes with agitation.
4. Transfer slide rack into dish Wash II. Incubate for 15 sec with agitation. Rock the
dish to move the wash over the tops of the arrays.
5. Transfer slide rack into dish Wash III. Incubate for 15 sec with agitation.
6. Remove slide rack from Wash III and spin dry in an array-drying unit
(NimbleGen Array-Go-Round) for 1 min.
7. Store the dry array in a dark desiccator. Proceed immediately to the protocol for
One-color Scanning of NimlbeGen Arrays.
Component and Supplier List:
Component
Superscript™ DoubleStranded cDNA synthesis
kit
Superscript™ DoubleStranded cDNA synthesis
kit
RNase A Solution
Phenol:Chloroform:isoamyl
alcohol
7.5 M Ammonium Acetate
5mg/ml Glycogen
Phase Lock Tubes (light 1.5
mL)
Oligo dT Primer
Vendor
Invitrogen
Package Size
10 reactions
Item Number
11917-010
Invitrogen
50 reactions
11917-020
Novagen
Ambion
1ml
100mL
70856-3
9730
Sigma Aldrich
Ambion
Fisher Scientific
1L
5 X 1ml
200 tubes
A-2706
9510
E0032 007 961
NimbleGen does not recommend a supplier; the customer is
NimbleGen 8-8
Random Primer
0.5 M EDTA
Absolute Ethanol
Water, Reagent Grade,
ACS, Nonsterile Type1
100mM dNTP’s
iM Tris HCL pH7.4
1M MgCl2
Β-Mercatoethanol
Klenow Fragment 3’-5’exo5M NaCl
Isopropanol
Cy3 9mer Wobble
Cy3 CPK6 48MER(see
below)
MAUI mixer SL
NimbleGen Hybridization
kit 40-CGH, ChIP, &
Expresison
NimbleGen Hybridization
Kit 40 Refill-CGH, ChIP, &
Expression.
encouraged to use a vendor that they are comfortable with.
NimbleGen does not recommend a supplier; the customer is
encouraged to use a vendor that they are comfortable with.
Sigma Aldrich
100ml
E7889
Sigma Aldrich
500ml
E7023
VWR
2.5 gal
RC915025
Invitrogen
Sigma Aldrich
Sigma Aldrich
Sigma Aldrich
NEB
Sigma Aldrich
Sigma Aldrich
TriLink
Biotechnologies
IDT
4 X 25umole
1L
100ml
25ml
50,000u/ml
250ml
500ml
50 O.D.
200 O.D
250nmole
Biomicro
NimbleGen
12 Lids
40Rxn
10287-018
T2663
M1028
M3148
M0212M
71386
I9516
N46-0001-50
N46-0001-200
Custom Oligo
synthesis
02-A008-03
KIT002-2
Nimble Gen
40Rxn
KIT005-2
Oligo Ordering Information:
Source: Integrated DNA Technologies (800-328-2661)
Scale: 250 nmole, HPLC purified
3’ Cy3 CPK6-48MER:
5’ TTC CTC TCG CTG TAA CCT TGA ATA ATC CTA TCA AAC AAC TCA
3’