|
| Status |
Public on Nov 08, 2023 |
| Title |
HEV-infected HepaRG (SAMPLE 10) |
| Sample type |
RNA |
| |
|
| Source name |
HepaRG
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepaRG
treatment: HEV-3f
time: 26 days
moi: MOI 10
|
| Treatment protocol |
Two days before IFN treatment, differentiation medium was replaced by proliferation medium. Cells were then treated overnight with 200 IU/ml of IFN-β1a (PBL Interferon Source, Piscataway, NJ, USA).
Two days before infection, differentiation medium was replaced by proliferation medium. Cells were infected overnight with an HEV-3f inoculum diluted in proliferation medium to a final volume of 1ml. The viral suspension was then removed and cells were washed three times in PBS before adding 2 ml of proliferation medium. Every 2 to 3 days, one-half (1 ml) of the culture medium was replaced with fresh growth medium and infection maintained for up to 100 days. A HEV-3f strain originating from a French patient suffering from acute autochthonous hepatitis E was used (GenBank under accession number JN906974)
|
| Growth protocol |
HepaRG were seeded into 6- well plates and cultured in proliferation medium for 2 weeks. Medium was then replaced for 2 extra weeks by “differentiation medium” supplemented with 1.2% DMSO (Sigma-Aldrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy Mini Kit including a DNase I treatment step.
|
| Label |
SYBR Green
|
| Label protocol |
PCR assays were performed using the Human Type I Interferon Response PCR array (Qiagen) following the Manufacturer’s instructions. Reverse transcription was performed from 500 ng total RNA using the RT2 First Strand Kit (Qiagen). Quantitative real-time PCR were performed (Light Cycler 96, Roche) with 45 cycles at 95 oC for 15 seconds and 60 oC for 30 seconds.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Test
|
| Data processing |
The normalization and all the data analysis were performed according to the manufacturers instructions using their web-based software package GeneGlobe Data Analysis Center : https://geneglobe.qiagen.com/fr/analyze
For the normalization it uses the average of four housekeeping genes: ACTB, GAPDH, HSP90AB1 and GUSB
Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ave Ct_HKG)]
The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data.
Matrix normalized worksheet reports normalized signal (against housekeeping genes).
Fold Change worksheet reports test/control (i.e., HEV-3f/Mock) ratios.
|
| |
|
| Submission date |
Sep 22, 2023 |
| Last update date |
Nov 08, 2023 |
| Contact name |
Virginie Doceul |
| E-mail(s) |
virginie.doceul@vet-alfort.fr
|
| Organization name |
UMR VIROLOGIE INRAE-ANSES-ENVA
|
| Street address |
7 avenue du Général de Gaulle
|
| City |
Maisons-Alfort |
| ZIP/Postal code |
94700 |
| Country |
France |
| |
|
| Platform ID |
GPL33776 |
| Series (2) |
| GSE243855 |
Real-time quantitative PCR analysis of human HepaRG cells infected with hepatitis E virus (HEV) [pre-array] |
| GSE243864 |
Real-time quantitative PCR analysis of liver tissue from pigs infected experimentally with hepatitis E virus (HEV) |
|
