|
| Status |
Public on Dec 01, 2023 |
| Title |
500 μM_oleic_acid_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
500 μM oleic acid, HepG2
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: HepG2
treatment: 500 uM oleic acid
|
| Treatment protocol |
HepG2 cells were seeded at 1 × 105 cells/well in 6-well plates and incubated at 37℃ under 5% CO2. The culture medium was replaced with 0, 50, 200, or 500 μM oleic acid after 72 h and was incubated for 24 h.
|
| Growth protocol |
In vitro adipogenesis used the liver cancer cell line, HepG2 cells. The Japanese Collection of Research Bioresources provided HepG2 cells (JCRB1054), and Dulbecco’s Modified Eagle Medium (Fujifilm wako) with 10% fetal bovine serum and antibiotics, including penicillin and streptomycin, cultured HepG2 cells. Cultures cultivated at 37°C under 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using the RNeasy Mini kit (QIAGEN) following the manufacturer’s instructions. A NanoDrop spectrophotometer (NanoDrop Technologies) assessed the quality and concentration of total RNAs. All RNA samples demonstrated 260/280-nm absorbance ratios of 1.8–2.0. An Agilent 2100 Bioanalyzer and an Agilent RNA 6000 Pico Kit (Agilent Technologies) confirmed the peaks of total RNAs, following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine 3 (Cy3)-labeled cRNA was synthesized from 150 ng of total RNA using a Low Input Quick Amp Labeling Kit (Agilent Technologies), according to the manufacturer’s instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
Cy3-labeled cRNA (600 ng) was fragmented at 60°C for 30 minutes in a reaction volume of 25 μL containing 1× Agilent fragmentation buffer and 2× Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 μL of 2× Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE microarray 8×60K (G4851C) (Agilent Technologies) for 17 hours at 65°C in a rotating hybridization oven (Agilent Technologies). After hybridization, microarrays were washed at room temperature for 1 min with GE Wash Buffer 1 (Agilent Technologies) and at 37°C for 1 min with GE Wash buffer 2 (Agilent Technologies), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the SureScan Microarray Scanner (G4900DA) (Agilent Technologies) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 2 μm high sensitivity, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent Technologies) using default parameters (protocol GE1_1105_Oct12 and Grid: 039494_D_F_20200924) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
The obtained data were normalized by quantile normalization using GeneSpring GX14.9 software (Agilent Technologies). In addition, the minimum expression value (raw signal) was set at 100 to filter by expression level. GeneSpring GX14.9 software was used to obtain expression data with more than 2.0-fold expression change in each treated group (50, 200, and 500 μM) compared to the control group (0 μM). Matrices were denoted with gProcessedSignal (raw).
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| |
|
| Submission date |
Nov 17, 2023 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Mitsuru Chiba |
| E-mail(s) |
mchiba32@hirosaki-u.ac.jp
|
| Phone |
+81172395965
|
| Organization name |
Hirosaki University
|
| Department |
Graduate School of Health Sciences
|
| Lab |
Department of Bioscience and Laboratory Medicine
|
| Street address |
66-1 Hon-cho
|
| City |
Hirosaki |
| State/province |
Aomori |
| ZIP/Postal code |
036-8564 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE248166 |
Analysis of gene expression changes in lipid droplet formation of human liver cancer cell HepG2 |
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