|
| Status |
Public on Aug 08, 2008 |
| Title |
Gene expression profiles of HL-60 cells exposed to toluene (IC50) [sample 2] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HL-60 cells treated with DMSO for 3 h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HL-60
cell type: human promyelocytic leukemia cells
treatment: DMSO
treatment time: 3 h
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the HL-60 cells treated to DMSO for 3 h using the Trizol reagent (Invitrogen, USA) and purified using RNeasy mini kit (Qiagen, USA) according to the manufacturer's instructions. Genomic DNA was removed using RNase-free DNase set (Qiagen, USA) during RNA purification. RNA quality were assessed with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) (260/280 nm ratios) and only samples with an A260/A280 ratio between 1.9 and 2.2 were considered for suitable use. The RNA quality was checked with the Experion automated electrophoresis station (Bio-Rad Laboratories, USA) (28S/18S ratio).
|
| Label |
Cy3
|
| Label protocol |
Gene expression analysis was conducted on the RNA samples using 35 K whole human genome microarray (Operon Biotechnologies, Inc. Germany). Triplicate analysis was performed for each chemical, simultaneously. Labeling and hybridization were performed by instruction of Platinum Biochip Reagent Kit (GenoCheck Co. Ltd., Korea). Cy3 dye for the controls (DMSO).
|
| |
|
| Channel 2 |
| Source name |
HL-60 cells treated with toluene (IC50) for 3 h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HL-60
cell type: human promyelocytic leukemia cells
treament: toluene
treament dose: IC50
treament time: 3 hr
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the HL-60 cells treated to IC50 dose of toluene for 3 h, using the Trizol reagent (Invitrogen, USA) and purified using RNeasy mini kit (Qiagen, USA) according to the manufacturer's instructions. Genomic DNA was removed using RNase-free DNase set (Qiagen, USA) during RNA purification. RNA quality were assessed with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) (260/280 nm ratios) and only samples with an A260/A280 ratio between 1.9 and 2.2 were considered for suitable use. The RNA quality was checked with the Experion automated electrophoresis station (Bio-Rad Laboratories, USA) (28S/18S ratio).
|
| Label |
Cy5
|
| Label protocol |
Gene expression analysis was conducted on the RNA samples using 35 K whole human genome microarray (Operon Biotechnologies, Inc. Germany). Triplicate analysis was performed for each chemical, simultaneously. Labeling and hybridization were performed by instruction of Platinum Biochip Reagent Kit (GenoCheck Co. Ltd., Korea). Cy5 dye for the treated samples.
|
| |
|
| |
| Hybridization protocol |
Labeling and hybridization were performed by instruction of Platinum Biochip Reagent Kit (GenoCheck Co. Ltd., Korea). This was followed by the coupling of the Cy3 dye for the controls (DMSO) or Cy5 dye for the treated samples. Hybridization was performed in a hybridization oven at 62℃ for 12 h. After washing (2× SSC/0.1% SDS for 2 min at 58℃, 1× SSC for 2 min at RT and 0.2× SSC for 3 min at RT), the slide was dried by centrifugation at 800 rpm for 3 min at RT.
|
| Scan protocol |
Hybridization images on the slides were scanned by ScanArray Lite (PerkinElmer Life Sciences, USA). Scanned images were analyzed with GenePix 3.0 software (Axon Instruments, USA) to obtain gene expression ratios.
|
| Description |
HL-60 cells were treated with 2.74 mM toluene for 3 h, and the RNA was subjected to microarray analysis.
|
| Data processing |
The fluorescent intensity of each spot was calculated by local median background subtraction. The robust scatter-plot smoother LOWESS function was used to perform intensity dependent normalization for the gene expression. Scatter plot analysis was made by Microsoft Excel 2003. Significance Analysis of Microarray (SAM) was performed for the selection of the genes with significant gene expression changes (Tusher et al., 2001). Computing a p-value for each gene assessed the statistical significance of the differential expression of genes. To determine the p-value, a permutation procedure was used and for each permutation, two-sample t statistics were computed for each gene. Genes were considered differentially expressed when logarithmic gene expression ratios in three independent hybridizations were more than 1.5 or less than 0.66, i.e., 1.5-fold difference in expression level, and when the p-values were less than 5.
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| |
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| Submission date |
Aug 06, 2008 |
| Last update date |
Feb 24, 2010 |
| 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 |
| |
|
| Platform ID |
GPL7080 |
| Series (2) |
| GSE12353 |
Gene expression profiles of human promyelocytic leukemia cell lines exposed to six volatile organic compounds |
| GSE24899 |
Differential gene expression profiles of human promyelocytic leukemia cell line exposed to benzene toluene, and o-xylene |
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