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Status |
Public on Nov 08, 2023 |
Title |
HEV-infected HepaRG (SAMPLE 11), D100 plaque 1 |
Sample type |
RNA |
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Source name |
HepaRG
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Organism |
Homo sapiens |
Characteristics |
cell line: HepaRG treatment: HEV-3f time: 100 days moi: MOI 100 experiment: 2
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Treatment protocol |
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 100 days. A HEV-3f strain originating from a French patient suffering from acute autochthonous hepatitis E was used (GenBank under accession number JN906974)
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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).
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted with RNeasy Mini Kit including a DNase I treatment step.
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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.
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Hybridization protocol |
n/a
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Scan protocol |
n/a
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Description |
Test
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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.
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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 |
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Platform ID |
GPL33776 |
Series (2) |
GSE243856 |
Real-time quantitative PCR analysis of human HepaRG cells infected with hepatitis E virus (HEV) [D100 plaque 1] |
GSE243864 |
Real-time quantitative PCR analysis of liver tissue from pigs infected experimentally with hepatitis E virus (HEV) |
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