726839334
NimbleGen double stranded cDNA synthesis and Labeling
(For total RNA)
Ambion MessageAmp II aRNA Amplification Kit, Catologue #AM1753, and NimbleChip Arrays User’s Guide
reagents are used for this protocol.
I. Oligo-dT Prime RNA
Reagent
Final Quantity in Rxn
Oligo-dT
totRNA
Water (RNAse free)
1ul
2ug
Volume
1ul
maximum up to 11ul
bring volume up to 12ul
Final Volume = 12 ul
 Incubate RNA/Oligo Mixture @ 70˚C - 10 min in a PCR tube.
 Quick Chill on ice (immediately).
II. First Strand cDNA Synthesis
Reagent
10X First Strand Buffer
dNTP Mix
RNase Inhibitor
ArrayScript
Volume for 1 Rxn
2ul
4ul
1ul
1ul
Final Volume = 8 ul
 Add 8 ul Rxn mix to Oligo/RNA mixture.
 Incubate @ 42°C – 2 hrs.
III. Second Strand cDNA Synthesis
Reagent
Water (RNAse free)
10X Second Strand Buffer
dNTP Mix
DNA Polymerase
RNase H
Volume for 1 Rxn
63ul
10ul
4ul
2ul
1ul
Final Volume = 80 ul
 Add 80 ul Rxn mix to ss-cDNA mixture.
 Incubate @ 16°C – 2 hrs.
 Store @ -20°C or Purify cDNA.
IV. cDNA Purification
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Pre-heat nuclease-free water to 50°C.
Add 250ul cDNA Binding Buffer to each cDNA sample.
Apply mixture to center of cDNA Filter Cartridge.
Spin @ 10,000 x g – 2 min.
Decant flo-thru.
Wash: Refill original container with 500ul Wash Buffer.
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Spin @ 10,000 x g – 2 min.
Decant flo-thru.
Spin @ 10,000 x g – 2 min to dry cDNA Filter.
Transfer cDNA Filter to a new 1.5ml tube.
Elute cDNA by adding 9ul of 50°C nuclease-free water.
Stand at room temp – 2 min.
Spin @ 10,000 x g – 2 min.
Repeat column elution.
Keep on ice or store @ -20°C.
V. IVT to Synthesize aRNA
Reagent
Volume for 1 Rxn
25mM ATP, CTP, GTP Mix
50mM UTP Solution
T7 10X Reaction Buffer
T7 Enzyme Mix
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12ul
6ul
4ul
4ul
Final Volume = 26 ul
Add 26 ul Rxn mix to ds-cDNA in a new PCR tube.
Incubate @ 37°C – 14 hrs.
Add 60ul nuclease-free water to stop rxn.
Store @ -20°C or Purify aRNA.
VI. aRNA Purification (to be performed in a vacuum hood)
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Pre-heat nuclease-free water to 50°C.
Add aRNA sample to a new 1.5ml tube.
Add 350ul aRNA Binding Solution to each aRNA sample.
Immediately Add 250ul ACS grade 100% EtOH to each aRNA sample.
Immediately Mix by pipet, DO NOT VORTEX
Immediately Apply mixture to center of an aRNA Filter Cartridge.
Spin @ 10,000 x g – 2 min.
Decant flo-thru.
Wash: Refill original container with 650ul Wash Buffer.
Spin @ 10,000 x g – 2 min.
Decant flo-thru.
Repeat Wash.
Spin @ 10,000 x g – 2 min to dry aRNA Filter.
Transfer aRNA Filter to a new 1.5ml tube.
Elute aRNA by adding 50ul of 50°C nuclease-free water.
Stand at room temp – 2 min.
Spin @ 10,000 x g – 2 min.
Repeat column elution.
QC samples: NanoDrop and bioanalyze.
In order to proceed Samples must be a concentration of 0.4ug/ul, 260/280>1.8, and 260/230>1.7.
Store @ -20°C or Synthesize 2nd Round ss-cDNA.
VII. Second Round Prime RNA
Reagent
Second Round Primer
aRNA
Final Quantity in Rxn
2ul
4ug
Volume
2ul
maximum up to 10ul
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Water (RNAse free)
bring volume up to 12ul
Final Volume = 12 ul
 Incubate RNA/Primer Mixture @ 70˚C - 10 min in a PCR tube.
 Quick Chill on ice (immediately).
VIII. Second Round First Strand cDNA Synthesis
Reagent
10X First Strand Buffer
dNTP Mix
RNase Inhibitor
ArrayScript
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Volume for 1 Rxn
2ul
4ul
1ul
1ul
Final Volume = 8 ul
Add 8 ul Rxn mix to Primer/RNA mixture.
Incubate @ 42°C – 2 hrs.
Add 1 ul RNase H to each sample.
Incubate @ 37°C – 30 min.
IX. Second Round Oligo-dT Prime RNA
 Add 5ul Oligo-dT Primer
 Incubate RNA/Oligo Mixture @ 70˚C - 10 min in a PCR tube.
 Quick Chill on ice (immediately).
X. Second Round Second Strand cDNA Synthesis
Reagent
Water (RNAse free)
10X Second Strand Buffer
dNTP Mix
DNA Polymerase
Volume for 1 Rxn
58ul
10ul
4ul
2ul
Final Volume = 74 ul
 Add 74 ul Rxn mix to ss-cDNA mixture.
 Incubate @ 16°C – 2 hrs.
 Store @ -20°C or Purify ds-cDNA.
XI. Second Round ds-cDNA Purification
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Pre-heat nuclease-free water to 50°C.
Add 250ul cDNA Binding Buffer to each cDNA sample.
Apply mixture to center of cDNA Filter Cartridge.
Spin @ 10,000 x g – 2 min.
Decant flo-thru.
Wash: Refill original container with 500ul Wash Buffer.
Spin @ 10,000 x g – 2 min.
Decant flo-thru.
Spin @ 10,000 x g – 2 min to dry cDNA Filter.
Transfer cDNA Filter to a new 1.5ml tube.
Elute cDNA by adding 11ul of 50°C nuclease-free water.
Stand at room temp – 2 min.
Spin @ 10,000 x g – 2 min.
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Repeat column elution.
QC samples: NanoDrop and bioanalyze.
In order to proceed Samples should be a concentration of 0.1ug/ul, 260/280>1.8, and 260/230>1.7.
Keep on ice or store @ -20°C.
XII. Cy3 Prime ds-cDNA
Reagent
Final Quantity in Rxn
Cy-3 labeled 9mers
ds-cDNA
Water (RNAse free)
40ul
2ug
Volume
40ul
maximum up to 40ul
bring volume up to 80ul
Final Volume = 80 ul
 Incubate ds-cDNA/Cy3 9mer Mixture @ 98˚C - 10 min in a PCR tube.
 Quick Chill on ice water bath 10 min (immediately).
XIII. ds-cDNA Cy3 Labeling
Reagent
(DO NOT VORTEX!!!)
Volume for 1 Rxn
50X dNTP Mix
Water (RNAse free)
Klenow (50U/ul)
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10ul
8ul
2ul
Final Volume = 20 ul
Add 20 ul Rxn mix to Primer/RNA mixture.
Mix by pipetting up and down 10 times. DO NOT VORTEX!!!
Incubate @ 37°C – 2 hrs.
Add 10 ul 0.5M EDTA to each sample to stop reaction.
Add 11.5 ul 5M NaCl to each sample.
Vortex samples and quick spin.
XIV. ds-cDNA Cy3 Labeled Purification
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Add labeled sample to a new 1.5ml tube containing 110 ul Isopropanol.
Incubate @ room temp – 10 min in the dark.
Spin @ 12,000 x g – 10 min.
Remove supernatant with a pipet.
Rinse pellet with 500ul 80% ice-cold EtOH. Dislodge pellet form tube wall.
Spin @ 12,000 x g – 2 min.
Remove supernatant with a pipet.
Dry contents in a SpeedVac 5 min.
Rehydrate pellet in 25 ul RNase free water. Vortex.
QC samples: NanoDrop and bioanalyze.
Calculate appropriate amount of sample for array and mixer.
Cy3-labeled cDNA
Prokaryotic
Eukaryotic
Prokaryotic
Eukaryotic
Array Type
1-Plex
1-Plex
4-Plex
4-Plex
Mixer
SL
SL
X4
X4
Quantity
6.5ug
13ug
2ug
4ug
 Dry contents in a SpeedVac.
 Keep on ice or store @ -20°C.
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726839334
Reagents/Supplies for NimbleGen Labeling Protocol
Description
Amino Allyl Message Amp II Kit
100mM dNTPs
5’ Cy3-labeled Random Nonamers (9mer “Wobble”)
B-Mercaptoethanol
1M MgCl2
1M Tris HCl, pH7.4
Klenow Fragment 3’->5’ exo- (50U/ul)
0.5M EDTA
5M NaCl
Isopropanol
Absolute Ethanol
RNA 6000 Nano Kit (x3/rxn)
Catalog#
Vendor
Unit
Cost
AM1753
10297-018
N46-0001-50
M3148-25ML
M-1028-100ML
T-2663-1L
M0212M
E-7889-100ML
71386-250ML
I-9516-25ML
E702-3-500ML
5067-1511
Ambion
Gibco/Stockroom
TriLink Biotech
Sigma Aldrich
Sigma Aldrich
Sigma Aldrich
NEB
Sigma Aldrich
Sigma Aldrich
Sigma Aldrich
Sigma Aldrich
Agilent
1085.00
143.20
975.00
10.90
43.40
33.60
224.00
37.10
15.50
10.70
41.00
485.00
#rxns/unit
Cost/rxn
10
2000
50
312,500
190,476
190
25
9,090
21,739
227
1250
300
108.50
0.07
19.50
0.01
0.01
0.18
8.96
0.01
0.01
0.07
0.04
1.62
Total Reagent Cost Per Rxn
142.22
Solutions Needed for NimbleGen ds-cDNA Synthesis & Labeling Protocol
Solution
10X TE
50X dNTP Mix
Random 9mer Buffer
Cy3-labeled 9mers
Reagents
1.5ml 1M Tris HCl, pH7.4
0.3ml 0.5M EDTA
13.2ml nuclease free water
250ul nuclease free water
50ul 10XTE
50ul dATP
50ul dGTP
50ul dTTP
50ul dCTP
8.6 ml nuclease free water
1.25ml 1M Tris HCl, pH7.4
125ul 1M MgCl2
17.5ul B-Mercaptoethanol
Cy3-labeled 9mer Wobble Primers
Random 9mer Buffer
Preparation
Mix
Storage
Room Temp
Mix and aliquot into single-use
amounts.
-20C
Mix. Prepare fresh each time
primers are diluted.
Do not store.
Dilute Cy3-labeled 9mers to 1
O.D./42ul Random 9mer Buffer.
Aliquot into 40ul individual
reaction volumes in thin-walled
PCR tubes.
-20C protected
from light.
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