|
| Status |
Public on Dec 10, 2011 |
| Title |
neural cell_virus control_Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
EV71-infected cells without treatment with Hep
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SK-N-SH
tissue: Human Caucasian neuroblastoma
morphology: Epithelial
|
| Treatment protocol |
SK-N-SH cells were seeded at 1.3 × 105 cells/1600 in 12-well plates and incubated at 37ºC/5% CO2 for two days. After reaching 80% cell confluency, the media was aspirated followed by addition of 1600 ul of each of the following conditions: cell control, virus control at 100 TCID50, compound control, and treatment (Hep-treated cells infected with 100 TCID50 of EV71), each in three wells (two wells for antiviral or cytotoxicity assays, one well for RNA isolation). Each experiment was independently replicated three times. Following incubation at 37ºC/5% CO2 for a further 48 hours, antiviral and cytotoxicity assays were quantified by MTT. At the same time, total RNA was isolated from allocated wells.
|
| Growth protocol |
SK-N-SH cells were maintained in Dulbecco’s Modified Eagle’s Medium with high glucose supplemented with 10% heat inactivated Fetal Bovine Serum. The cells were not passaged more than 20 times after reviving from the original stock. A low passage clinical isolate of EV71 (the isolate number 99018233, from Victorian Infectious Disease Reference Laboratories) was propagated in 80% confluent monolayers of Vero cells using serum-free DMEM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was isolated and purified from SK-N-SH cells of each of the four conditions using the RNeasy Mini Kit of Qiagene,
|
| Label |
biotin
|
| Label protocol |
The Affymetrix GeneChip WT Terminal Labeling Kit (PN 900671 or equivalent) is used for the fragmentation and labeling of the cDNA
|
| |
|
| Hybridization protocol |
Hybridization was performed into hybridization oven at 45±C, 60 rpm for 17 ± 1 hour followed by washing and scanning the arrays
|
| Scan protocol |
1. Clean glass from chip, put tape on septa and place chip in the autoloader 2. Start Scanner 3. After Scan, check controls at corners and grid alignment 4. Transfer data out of AGCC as .DTT files 5. Run .CEL files through Expression Console to generate .CHP files for QC 6. Backup all data to DVD, including flat files and DTT for archiving
|
| Description |
Gene expression data from EV71-infected cells not treated with Hep at 72 hours post infection
|
| Data processing |
Since Affymetrix does not put mismatch probes in the newer arrays such as GeneChip Human Gene 1.0 ST array, background noise was handled by a background-correction step during the normalization. Partek Genomics Suite version 6.5 was utilized to import and analyze raw data from the array cells and also import latest version of annotation from Affymetrix website (as of 8/8/2011). Using one-step Tukey’s bi-weight, normalized values of multiple probes for the same gene (at each replication) were summarized into a single value representing consensus level of expression for that gene. Then, the following five contrasts were made: Hep vs. CC; VC vs. CC; Cyto vs CC; Hep vs. VC; and Hep vs. Cyto. For each contrast, only samples from the two target groups were included. A single factor ANOVA inside Partek was applied to generate P values for each gene. Then, these P values were adjusted by Benjamini and Hochberg’s Multiple testing correction to reduce false discovery rate (FDR).
|
| |
|
| Submission date |
Dec 07, 2011 |
| Last update date |
Dec 10, 2011 |
| Contact name |
Hamid Reza Pourianfar |
| E-mail(s) |
[email protected]
|
| Phone |
0098-9365980932
|
| Organization name |
Current Address: Iranian Academic Centre for Education, Culture and Research, Mashhad Branch
|
| Department |
-
|
| Lab |
SWinburne University of Technology, Life Sciences,PC2-virology
|
| Street address |
Azadi Square
|
| City |
Mashhad |
| State/province |
Khorasan Razavi |
| ZIP/Postal code |
P.O.Box: 91775-1376 |
| Country |
Iran |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE34234 |
Microarrays analysis of anti-Enterovirus 71 activity of Heparin |
|
