|
| Status |
Public on Nov 17, 2014 |
| Title |
R_H001 |
| Sample type |
RNA |
| |
|
| Source name |
Human THP-1 cells
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: treatment with DMSO
|
| Treatment protocol |
Human THP-1 cells were pretreated with vehicle or PBHA (10 μM) before LPS stimulation (50 ng/ml). After 8 hours, the total cellular RNAs were manipulated for microarray assay.
|
| Growth protocol |
Human monocytic cells (THP-1, ATCC) were cultured in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 microg/ml streptomycin and 18 mM HEPES, pH 7.4. Cells were used at 90% confluence, and following treatments were handled in the same medium with 0.5% FBS (Chou et al., 2011).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction by RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNA purity was checked by optical density of NanoDrop ND-1000 and agarose electrophoresis with the result of A260/A280≧1.8, no gDNA contamination. RNA integrity was measured by Agilent RNA 6000 Nano Assay (RIN≧8).
|
| Label |
Cy5
|
| Label protocol |
1 µg of total RNA was reverse-transcribed and amplified using Amino Allyl aRNA Amplification Kit (Phalanx Biotech Group, Taiwan) and Cy5 dyes (Amersham Pharmacia, Piscataway, NJ, USA).
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| |
|
| Hybridization protocol |
Fluorescent targets were hybridized to the Human Whole Genome OneArray® with Phalanx hybridization buffer using Phalanx Hybridization System. After 16 hrs hybridization at 50 ℃, non-specific binding targets were washed away by three different washing steps (WashⅠ 42 ℃ 5 mins；Wash Ⅱ 42 ℃, 5 mins, 25 ℃ 5 mins; Wash Ⅲ rinse 20 times)
|
| Scan protocol |
The arrays were scanned by G2505C (ScanPower=100 n/a, PMTGain=0 n/a) and quantify the fluoresence intensity.
|
| Data processing |
The signal intensity of each spot was loaded into Rosetta Resolver System® (Rosetta Biosoftware) to process data analysis. The error model of Rosetta Resolver System® could remove both systematic and random errors form the data. We filtered out spots that the flag is less than 0. Spots that passed the criteria were normalized by 50% media scaling normalization method.Normalize intensities: Median scaling performed on data set without flagged and control data. Merge technical replicate data: Average intensity values calculated on technical replicates.
Calculate average ratios of the hybridization replicates and find significant values for each gene by pairwise comparision (Ranking and hypothesis testing).
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| |
|
| Submission date |
Nov 14, 2013 |
| Last update date |
Nov 17, 2014 |
| Contact name |
George Hsiao |
| E-mail(s) |
geohsiao@tmu.edu.tw
|
| Phone |
+886-2-27374622
|
| Organization name |
Taipei Medical University
|
| Department |
Pharmacology
|
| Street address |
No. 250, Wu-Hsing Str
|
| City |
Taipei |
| ZIP/Postal code |
110 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL16951 |
| Series (1) |
| GSE52365 |
Microarray analysis of gene expression profiles in PBHA-treated THP-1 cells after LPS stimulation |
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