|
| Status |
Public on Apr 23, 2013 |
| Title |
1h_5mMHMBA_rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HD-MEL_at 1h after 5mM HMBA treatment
|
| Organism |
Mus musculus |
| Characteristics |
cell line: T-3-Cl-2-0.fl (Riken Cell No. RCB0561)
cell type: High differentiation-inducible murine erythroleukemia cells
time point: at 1h after 5mM HMBA treatment
|
| Treatment protocol |
MEL cells were washed in PBS, and stored at -80 °C.
|
| Growth protocol |
MEL cells were cultured in RPMI1640 medium with 10% fetal calf serum at 37 °C with 5% CO2 concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of MEL cells was extracted using RNeasy Mini Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Twenty ug of the total RNA was labeled with amino-modified dNTP using a SuperScript Indirect cDNA Labeling System (Invitrogen). The amino-modified cDNA was purified with a S.N.A.P column (Invitrogen) and ethanol precipitation. The purified cDNA was labeled with Cye3 Mono-Reactive Dye Pack (Amersham Biosciences) or Cye5 Mono-Reactive Dye Pack (Amersham Biosciences) by coupling reaction. The labeled cDNA was purified with a QIAquick PCR Purification Kit (Qiagen).
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| |
|
| Channel 2 |
| Source name |
HD-MEL_before chemical treatment
|
| Organism |
Mus musculus |
| Characteristics |
cell line: T-3-Cl-2-0.fl (Riken Cell No. RCB0561)
cell type: High differentiation-inducible murine erythroleukemia cells
time point: before chemical treatment
|
| Treatment protocol |
MEL cells were washed in PBS, and stored at -80 °C.
|
| Growth protocol |
MEL cells were cultured in RPMI1640 medium with 10% fetal calf serum at 37 °C with 5% CO2 concentration.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of MEL cells was extracted using RNeasy Mini Kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Twenty ug of the total RNA was labeled with amino-modified dNTP using a SuperScript Indirect cDNA Labeling System (Invitrogen). The amino-modified cDNA was purified with a S.N.A.P column (Invitrogen) and ethanol precipitation. The purified cDNA was labeled with Cye3 Mono-Reactive Dye Pack (Amersham Biosciences) or Cye5 Mono-Reactive Dye Pack (Amersham Biosciences) by coupling reaction. The labeled cDNA was purified with a QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
cDNA microarrays with varied spotting patterns (with the same total number of cDNA clones) were used. For HMBA and TSA experiments, 22k cDNAs were separately spotted on two glass slides for one analysis, clone ID H3001A01 to H3120H12 (approx. 11k cDNA clones) were spotted on one slide, and H3121A11 to H4079G07 (approx. 11k cDNA clones) were spotted on the other slide. For DMSO experiment, 22k cDNAs were separately spotted on two glass slides for one analysis, clone ID H3001A01 to H3159G07 (approx. 15k cDNA clones) were spotted on one slide, and H4001A11 to H4079G07 (approx. 7k cDNA clones) were spotted on the other slide. Therefore, raw data files have varied counts for data rows.
Microarray was pre-hybrized at 50°C, 60 minutes in 5 x saline sodium citrate buffer (SSC) contained 0.1% sodium dodecyl sulfate (SDS)and 0.1% bovine serum albumin (BSA), washed with water, and rinsed with isopropanol. Mixed Cy3 and Cy5 labeled cDNA solution was denaturation at 95°C, 5 minutes in 5 x SSC contained 1.67 mg/mL acetylated BSA. After cooling, SDS was added to final concentration 0.33%. The microarray was covered with a Gap cover glass (Matsunami), hybridization solution was injected, and the microarray was placed in a hybridization cassette (TeleChem). Hybridization was performed at 50°C for 14 hours. After hybridization, the microarray was washed using a Wash Station (TeleCham) as follow; at 42°C 5 minutes in 2 x SSC contained 0.1%SDS, at room temperature 10 minutes in 0.1 x SSC contained 0.1%SDS, and finally twice in 0.1 x SSC at room temperature for 2min. The microarray was dried by centrifugation.
|
| Scan protocol |
Hybridization images were scanned using a ScanArray 5000(Perkin-Elmer). Fluorescence intensity of the microarray was quantified using QuantArray 3.0 software (Perkin-Elmer).
|
| Description |
Technical replicate 4 of 4
The sample-dye assignment applies to both raw data files.
|
| Data processing |
The microarray data was analyzed using GeneSpring 6.0 (Silicon Genetics). Intensity difference between Cy3 and Cy5 was normalized by LOWESS method. Signal intensities were higher than negative spot.
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| |
|
| Submission date |
Jan 29, 2013 |
| Last update date |
Apr 24, 2013 |
| Contact name |
Hitoshi Sasaki |
| E-mail(s) |
[email protected]
|
| Organization name |
Tokyo University of Science
|
| Department |
Department of Biological Science and Technology
|
| Lab |
Murakami Laboratory
|
| Street address |
2641 Yamazaki
|
| City |
Noda |
| State/province |
Chiba |
| ZIP/Postal code |
278-8510 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16035 |
| Series (1) |
| GSE43849 |
Expression profiles of high differentiation-inducible murine erhthroleukemia cells |
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