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Platform GPL10703 Query DataSets for GPL10703
Status Public on Jul 24, 2010
Title KIST custom metabolite Agilent 4x44 K array
Technology type in situ oligonucleotide
Distribution custom-commercial
Organism Homo sapiens
Manufacturer BioMicro Systems, Inc. 1290 West 2320 South, Suite D Salt Lake City UT 84119
Manufacture protocol Materials
MAUI Mixer A4 Hybridization Chambers (Cat No. 02-A008-25)
A4/LC Hinged Clamp Assembly (Cat No. 02-A004-00)
SL/A4/LC Assembly/Disassembly Jig (Cat No. 02-033-03)
Gasket Brayer (Cat No. 02-054-00)
MAUI Humid Cover (Cat No. 02-D0031-00)
M-100 Microman® Pipette (Cat No. 01-014-00)
CP-100 Pipette Tips (Cat No. 01-015-00)
Port Plugs (Cat No. 03-D0002-00)
Port Plug Insertion Tool (Cat No. 04-D0005-00)
Cotton Swabs
1.5 ml Eppendorf® Tubes
Hybridization Buffer (see recipe below)
250 μl Pipette Conditioning Solution (see recipe below- used only to eliminate air from the pipette tip prior to filling the
hybridization chamber and not for the actual hybridization)
Additional BioMicro Systems Instructions & Procedures
Replacing Bay Clamp Assemblies on MAUI Hybridization Systems (CDP-02)
MAUI Humid Cover for the MAUI Hybridization System (CDP-12)
MAUI Mixer Sealing Using Port Plugs (CDP-13)
MAUI Mixer-Slide Assembly Procedure (see MAUI Hybridization System User’s Guide) (CDP-14)
MAUI Mixer Filling and Sealing Procedure (see MAUI Hybridization System User’s Guide) (CDP-14)
Modifications to Agilent Two-Color Microarray-Based Gene Expression Analysis --
Quick Amp Labeling and Hybridization Protocol
Below is a list of key adaptations made to the Agilent gene expression array labeling (Quick Amp Labeling) and
hybridization protocol when using the MAUI Hybridization System and 4x44k CGH arrays.
Table 1: Protocol Differences
Agilent SureHyb® System MAUI Hybridization System
Labeled Target(s) - Dry using a SpeedVac®
Final Sample Volume
4 x 44K array 110 μl 25 μl
Hybridization Mixture No formamide Add formamide for a final
concentration of 16%
Hybridization Temperature 65ºC 55ºC
Hybridization Time
4 x 44K array 17 hours 17 hours
Total RNA labeling should be performed according to Agilent Quick Amp Labeling protocol. For the microarray
hybridization process, following removal of the unincorporated dyes it is necessary to reduce the volume of the labeled
target in order to load it into the ultra-low volume MAUI Mixers. The following modifications have been made to the
protocol of Agilent Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling, version 5.7, March
2008):
MAUI® Mixer A4 on Agilent® 4 x 44K Gene Expression Arrays
Following the purification of the labeled and amplified RNA (step 9, page 24).
1. Combine the appropriate purified Cy5 and Cy3 labeled RNA samples.
2. Dry combined samples using an appropriate speed vacuum under low heat and protect samples from light during the
process (e.g. using aluminum foil). This may take 20 minutes or longer depending on the degree of vacuum, the
sample volume and total number of samples being prepared.
3. Prepare the hybridization mixture according to the following recipe at room temperature.
Note: Make the appropriate volume of hybridization mixture based on the number of samples prepared.
Table 2: Hybridization Mixture Recipe
Component Volume
Nuclease-Free Water 80.6 μl
Agilent 10X GE Blocking Agent 25 μl
25x Fragmentation Buffer 4.4 μl
Final Hybridization Mixture Volume 110 μl
4. Add 11 μl of the above hybridization mixture to each dried sample in a 1.5 ml Eppendorf Tube. Completely
resuspend the labeled sample(s) by gently pipetting the hybridization mixture up and down several times (avoid
causing bubbles). Briefly spin.
5. Incubate the samples at 60ºC for exactly 30 minutes to fragment RNA.
6. Add 4 ul of high-quality de-ionized formamide and 10 μl of 2x GE Hybridization Buffer to each sample to stop the
fragmentation reaction and make a final total volume of 25 μl for each array. Mix well but gently by pipetting. To
avoid introducing bubbles, do not vortex. Please note that the final concentration of formamide is 16% and the final
concentration of Agilent 2x GE hybridization buffer in the final hybridization mixture is 0.8x (not 1x), which is
optimized specifically for the MAUI Hybridization system.
Final Volume = Chamber Volume + (5 to 10 μl).
Note: The chamber volumes may vary slightly from lot to lot of MAUI Mixers. The actual chamber volume for each MAUI
Mixer is indicated on the MAUI Mixer packaging.
7. Spin tubes (highest speed for 1 minute) to collect the buffer at the bottom of the tubes and load samples as soon as
possible.
Protocol
Prior to using Agilent microarrays with the MAUI Hybridization System you will need to replace the standard bay clamp
assemblies with A4/LC bay clamp assemblies (see Bay Clamp Replacement Instructions CDP-02).
1. Set the MAUI Hybridization System temperature to 55ºC.
2. Prepare 250 μl of Pipette Conditioning Solution in a 1.5 ml Eppendorf tube and warm to 55ºC. The purpose of this
solution is to eliminate air from the pipette tip prior to aspirating the hybridization mixture for injection into the MAUI
Mixer hybridization chamber.
Table 3: Pipette Conditioning Solution Recipe
Component Volume
Nuclease-Free Water 80.6 μl
Agilent 10X Blocking Agent 25 μl
25x Fragmentation Buffer 4.4 ul
Agilent 2x GE Hybridization Buffer 100 μl
High Quality De-Ionized Formamide 40 μl
Final Pipette Solution Volume 250 μl
- 3 -
CDP-20-REVA DEC 2008
3. Place the solution at 55ºC a few minutes before use.
4. Open a single MAUI Mixer A4. Attach the mixer to the Agilent microarray slide using an SL/A4/LC
Assembly/Disassembly jig, as described in the MAUI Hybridization System User’s Guide. Make sure the “active
array side” of the slide faces up and the barcode end slides into the jig first.
Note: Completely bray all portions of the adhesive gasket. The adhesive gasket will appear clear (not cloudy) when sealed
correctly. Do not use excessive pressure when braying directly over the microarray surface, as this may result in damage to
the array.
BioMicro Systems highly recommends that you practice assembling and loading MAUI Mixers with used Agilent microarrays
and Pipette Solution containing 1% Bromophenol Blue.
5. Fill a single chamber of the MAUI Mixer A4.
a. Set the M-100 Microman Pipette to 3μl more than the chamber volume indicated on the MAUI Mixer
package label.
b. Use a new CP-100 pipette tip and slowly pipette the pipette conditioning solution (step 2) up and down until
all air bubbles are removed from the tip.
c. With the pipette plunger depressed, transfer the pipette tip into the tube containing the hybridization
solution.
d. Slowly aspirate hybridization solution (being careful not to introduce bubbles into the tip).
e. Insert the pipette tip into the fill port of the desired chamber. Be sure to hold the pipette tip as vertically as
possible.
MAUI Mixer A4
f. With a continuous motion, slowly inject the hybridization solution into the chamber. After filling, DO NOT
wipe hybridization solution from fill and vent ports.
6. Seal the chamber using port plugs (see Port Plug Instructions).
a. Hold the star shaped port plug carrier to the side of the MAUI Mixer (not over it) with the raised marks on
the handle facing up.
b. Center a port plug over the fill port. You should be able to feel the end of the plug insert into the port.
c. Vertically center the port plug insertion tool over the aligned port plug. Using the tool, lightly (~ 2-4 lbs. of
pressure) push the port plug into the fill port. Tilt the tool slightly toward the carrier (away from the center of
the MAUI Mixer) and gently pull the carrier away from the tool. The edge of the port plug tool will cut/snap
the port plug off of the carrier.
d. Move the port plug tool back to a vertical position and push with just enough force to fully insert plug. Avoid
using excessive force, which may disrupt the gasket seal.
e. Slowly release the pressure on the port plug insertion tool to prevent air from being pulled into the vent port.
f. Seal the vent port in the same manner (always seal fill port first and then the vent port).
g. Use a cotton swab to blot excess buffer from around the sealed port plugs until the cotton swab appears
dry.
- 4 - CDP-20-REVA DEC 2008
h. Press lightly on the center
 
 
Contributor(s) Sarma SN, Kim Y, Kim J
Submission date Jul 20, 2010
Last update date Jan 17, 2013
Contact name Sailendra Nath Sarma
E-mail(s) sarma@kist.re.kr
Phone +8229585080
Fax +8229585059
Organization name Korea Institute of Science and Technology
Lab Toxicology
Street address P.O. Box 131, Cheongryang
City Seoul
ZIP/Postal code 136-791
Country South Korea
 
Samples (6) GSM567857, GSM567858, GSM567859, GSM567860, GSM567861, GSM567862
Series (1)
GSE23018 Differential gene expression profiles of human promyelocytic leukemia cell lines exposed to benzene and its metabolites

Data table header descriptions
ID
GB_ACC GenBank Accession Number
Updated_gene_name Gene name
Gene_symbol Symbol of gene
UniGene unigene number
Block Chip block
Column Chip column
Row Chip Row
SEQUENCE sequence

Data table
ID GB_ACC Updated_gene_name Gene_symbol UniGene Block Column Row SEQUENCE
20 NM_023929 zinc finger and BTB domain containing 10 ZBTB10 Hs.591868 1 20 1 TGACATGGTATTTGGTTTTGGGTATCTGTTACTTTGGCACTATTCTTGTTTGCCTCTTTA
22 NM_006292 tumor susceptibility gene 101 TSG101 Hs.523512 1 22 1 GTTGAGCTCTTCTTAAAGTATTCTTCTCTTCCTTTTATCAGTAGGTGCCCAGAATAAGTT
23 NM_015915 spastic paraplegia 3A (autosomal dominant) SPG3A Hs.584905 1 23 1 CTAGCCTATGCAATATGATAATGGGACTGACCCTTATCACCCTGTGCACTTGGGCATATA
24 NM_030971 sideroflexin 3 SFXN3 Hs.283844 1 24 1 ACTGACATTTCTTAGACATTCAGCAAAACCCCCACCTTAACCTCTTTTCTTTCTTGAGGG
25 NM_147777 sorting nexin 15 SNX15 Hs.80132 1 25 1 AGCGGCGAAAGGGGGCAGAGGACCTGCTTCGCTTCACTGTGCACATACCTGCGCTCAACA
26 NM_005957 "5,10-methylenetetrahydrofolate reductase (NADPH)" MTHFR Hs.214142 1 26 1 TAGAATCTTCTCTAAAACGTATTCTTTGTGGCATTCTGAAATAGGATTCATGATGATGCC
27 NM_013265 chromosome 11 open reading frame2 C11orf2 Hs.277517 1 27 1 ACTGCCACTTTCTGCAGCTCTACCTGTGGCGTTTTGTGGCCGACGAAGAACTCGTGCACT
28 NM_020683 adenosine A3 receptor ADORA3 Hs.281342 1 28 1 ACACGGGCTGGTACTGGTGTGGCATCCAGCGGGACTTTGCCAGGGATGACATGGATTTTA
30 BC036527 CTAGE family pseudogene CTAGEP Hs.620802 1 30 1 CCTGGCAATATTTTTGCTCTCTTCAAAAGTAATTCTGACTGATCTCATTTCAAGTAACTG
31 NM_005238 v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) ETS1 Hs.369438 1 31 1 ATGCCGGAAACTCTCCTTCTGTGAGTATAACTCCTGAGTTCCTATAGCAGATAAGATATA
33 NM_005652 telomeric repeat binding factor 2 TERF2 Hs.63335 1 33 1 TCCCTGGTAATCTGTAGAACCTTCTCCTAGGAAATGGTGAAGTCTATTAGGAGCCACTTG
50 NM_001012508 shadow of prion protein homolog (zebrafish) SPRN Hs.64968 1 17 2 AACATCCAAGGACAGGAGAGACATGGAGTGGCGCTGGACAGTCACGACAAGGACTTGCCT
51 BC036071 "ELAV (embryonic lethal, abnormal vision, Drosophila)-like 4 (Hu antigen D)" ELAVL4 Hs.213050 1 18 2 TGGATATAACACCAGATTTCAGTTCAGAGAACACTCGTTCAACATTCAGGGAAAGCTTTT
53 NM_005358 LIM domain 7 LMO7 Hs.207631 1 20 2 CTCCATACGAAAGCACTGTTGCAGATAGAAGAAGAGGTGGTTGCTGCTCATGTAGATCTA
54 NM_001760 cyclin D3 CCND3 Hs.534307 1 21 2 ATGCTGGCTTACTGGATGCTGGAGGTATGTGAGGAGCAGCGCTGTGAGGAGGAAGTCTTC
55 NM_002980 secretin receptor SCTR Hs.693596 1 22 2 GGGTGCTGGGACATCAATGCCAACGCATCCATCTGGTGGATCATTCGTGGTCCTGTGATC
57 NM_002047 glycyl-tRNA synthetase GARS Hs.404321 1 24 2 CAAAAGACATGATCAATGTGAAGAGATTCCAGAAAACACTATATGTGGAAGAAGTTGTTC
59 AB075502 chromosome 6 open reading frame 62 C6orf62 Hs.519930 1 26 2 TCCTTTGGAGTAAAACTAGTGCTTACCAGTTTCCAATTGTATTTAGCTTCTGGTTGGAAT
60 NM_198159 microphthalmia-associated transcription factor MITF Hs.166017 1 27 2 ACATACAGCAAGCCCAACGGCAGCAGGTAAAGCAGTACCTTTCTACCACTTTAGCAAATA
62 NM_013277 Rac GTPase activating protein 1 RACGAP1 Hs.505469 1 29 2 AAATTATTCTTAATGTCTGTAAAAACGATTTTCTTCTGTAGAATGTTTGACTTCATATTG

Total number of rows: 27507

Table truncated, full table size 3772 Kbytes.




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