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| Status |
Public on Jul 15, 2019 |
| Title |
Caco-2 after STEC Ec472 interaction, time 20 |
| Sample type |
RNA |
| |
|
| Source name |
Caco-2 cells, STEC interaction
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Caco-2
|
| Treatment protocol |
The Caco-2 cells were cultured in T flasks (25 cm2) and DMEM with FBS (10%) and penicillin-streptomycin (100 U/mL-100 μg/mL) in a 5% CO2 at 37oC. The cells were grown until confluence and the formation of a polarized epithelial cell monolayer. Twenty-four hours prior to interaction assays the cells were washed three times with 1X phosphate buffered saline and incubated with antibiotic-free DMEM containing 10% fetal bovine serum. Interaction assays were performed with 400 µL (Abs550=0.35) of bacterial culture placed on a Caco-2 monolayer. Subsequently, the cell cultures were incubated in 5% CO2 at 37oC for different time intervals. The time series - equally spaced times of 7.5 min up to 3 h – encompassed a total of 25 samples (T0 to T24). In T0 the Caco-2 cells were recovered just after bacterial inoculation. After enterocyte-STEC interaction, the cells were washed 5 times with 1X phosphate buffered saline. Subsequently, Caco-2 cells were lysed directly in the culture flasks with 600 µL of Buffer RLT (Qiagen, Valencia, CA) for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell lysate was collected and total RNA extraction was accomplished using the RNeasy Mini kit (Qiagen, Valencia, CA). RNA purity analysis and quantification was done using the NanoVue spectrophotometer. RNA quality was assessed on the Agilent BioAnalyzer 2100 (Agilent, Santa Clara, CA). All samples presenting RIN ≥ 7.0 were stored at -80°C until use in hybridization experiments.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the One-Color Microarray-Based Gene Expression Analysis - Quick Amp Labeling (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (Qiagen). RNA concentration and purity was determined by reading the absorbance at 260 and 280 nm on a spectrophotometer (NanoVue, GE Health Care, USA).
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4845A) for 17 hours at 65°C in a rotating Agilent hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Bundle using one color scan setting for 4x44k array slides.
|
| Description |
Gene expression from Caco-2 cells after interaction of STEC strain Ec472
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) and considering spots present none or only one flag (i.e. low intensity, saturation, controls, etc.). The selected transcripts were used for analysis using the R software and considering any NA per group and the expression values in Log (base 2).
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| |
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| Submission date |
Oct 02, 2017 |
| Last update date |
Jul 15, 2019 |
| Contact name |
Carlos Alberto Moreira-Filho |
| E-mail(s) |
carlos.moreira@hc.fm.usp.br
|
| Phone |
55-11-3061-8449
|
| Organization name |
Faculdade de Medicina da Universidade de Sao Paulo
|
| Department |
Departments of Pediatrics
|
| Lab |
LIM36
|
| Street address |
Av. Dr. Eneas Carvalho de Aguiar 647
|
| City |
Sao Paulo |
| State/province |
SP |
| ZIP/Postal code |
05403-900 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL13497 |
| Series (2) |
| GSE104488 |
Dynamic Gene Network Analysis of Caco-2 Cell Response to Shiga Toxin-Producing Escherichia coli-Associated Hemolytic–Uremic Syndrome |
| GSE104492 |
Dynamic gene network analysis of Caco-2 cell response to Shiga toxin-producing Escherichia coli-associated hemolytic-uremic syndrome |
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