|
| Status |
Public on Apr 23, 2013 |
| Title |
24h_HMBA_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LD-MEL cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Low differentiation-inducible murine erythroleukemia cells, A clone of T-3-Cl-2-0.fl.
treatment: HMBA
time: 24h
cell line: T-3-Cl-2-0.fl (Riken Cell No. RCB0561)
|
| 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 mg 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).
|
| |
|
| Channel 2 |
| Source name |
HD-MEL cells
|
| Organism |
Mus musculus |
| Characteristics |
treatment: HMBA
time: 24h
cell type: High differentiation-inducible murine erythroleukemia cells, A clone of T-3-Cl-2-0.fl.
cell line: T-3-Cl-2-0.fl (Riken Cell No. RCB0561)
|
| 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 mg 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 |
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 3 of 4. 24 hours after 3.0mM HMBA treatment.
control LD-MEL
target HD-MEL
|
| 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.
|
| |
|
| Submission date |
Sep 10, 2012 |
| 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) |
| GSE40754 |
Comparison of transcriptome of high differentiation-inducible murine erythroleukemia cells and low differentiation-inducible murine erythroleukemia cells |
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