|
| Status |
Public on Oct 03, 2019 |
| Title |
273M1 |
| Sample type |
RNA |
| |
|
| Source name |
Huh7 cell, with genotype B HBV HBsAg mutation A273G expression
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Huh7
cell type: hepatoma
transfectant: genotype B HBV HBsAg mutation A273G expression
|
| Treatment protocol |
Plasmid pEGFP-N1-HBVS-genoB/C encodes the wild-type envelope protein under the transcriptional control of CMV promoter. The plasmid pEGFP-N1-HBVS-genoB was used as the template for mutagenesis to create mutations T216C, A273G, and double mutant T216C/A273G at small surface (S) region, with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid pEGFP-N1-HBVS-genoC was used as the template for mutagenesis to create mutations A293G, C446G, A456G, A293G/C446G, C446G/A456G, A293G/A456G, and triple mutant A293G/C446G/A456G at small S region. All mutants were confirmed by DNA sequencing.
|
| Growth protocol |
Huh-7 human hepatoma cells were grown at 37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, under an atmosphere of 5% CO2. A total of 3 × 105 Huh7 cells per well were seeded in a six-well plate 24 hours prior to transfection. Wild-type or mutant envelope expression vectors were transfected into Huh7 cells by using lipofectamine 3000 (Thermo Fisher Scientific) according to the manual of the manufacturer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After transfection, the cells were washed with cold PBS and then cells were harvested. Total RNA was extracted from Huh7 cells using TRIzol reagent (Life Technologies, Grand Island, NY, USA) and chloroform extraction. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
0.2 μg of total RNA from transfected Huh7 cells was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 (CyDye, Agilent Technologies, USA) during the in vitro transcription process.
|
| |
|
| Hybridization protocol |
0.6 μg of Cy3-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes. Correspondingly fragmented labeled cRNA is then pooled and hybridized to Agilent SurePrint Microarray (Agilent Technologies, USA) at 65°C for 17 h. After washing and drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of Huh7 cells with genotype B HBV HBsAg mutation A273G expression
|
| Data processing |
Scanned images are analyzed by Feature extraction10.5.1.1 software (Agilent Technologies, USA), an image analysis and normalization software is used to quantify signal and background intensity for each feature. Raw signal data was normalized by quantile normalization for differential expressed genes discovering.
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| |
|
| Submission date |
Oct 11, 2016 |
| Last update date |
Oct 03, 2019 |
| Contact name |
Wen-Chun Liu |
| E-mail(s) |
[email protected]
|
| Organization name |
National Cheng Kung University
|
| Street address |
138 Sheng-Li Road
|
| City |
Tainan |
| ZIP/Postal code |
70403 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL21061 |
| Series (1) |
| GSE87804 |
The gene expression signatures for Huh7 cell lines with HCC-associated HBV variants in small S proteins transfection |
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