|
| Status |
Public on Jan 05, 2018 |
| Title |
Caco after 3 hrs interaction with STEC EC472, replicate 1 [EC472] |
| Sample type |
RNA |
| |
|
| Source name |
Caco-2 cell, STEC interaction
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: intestinal cell line Caco-2
stress: interaction with STEC strain EC472
|
| Treatment protocol |
The Caco-2 intestinal cells were maintained in DMEM medium with 10% fetal bovine serum (complete medium) and penicillin-streptomycin in a 5% CO2 incubator at 37°C until cells differentiate in enterocyte-like. Prior to interaction assays Caco-2 cells were washed and incubated with complete medium. After 24 hrs 1/10 volume of bacterial culture were placed on a Caco-2 monolayer and incubated in a 5% CO2 at 37°C for 3 hrs. After this period Caco-2 cells infected and uninfected (control) were washed 3 times with phosphate buffered saline (PBS) and the cells were recovered with 5 mL of PBS by pipetting. The cells were then centrifuged for 5 min at 5000 xg and the pellet was ressuspended in 600 µL of RNAlater Reagent (Qiagen cat. no. 76154, Valencia, CA) for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells after lysing with RLT buffer and the RNeasy Mini Kit (Qiagen cat no. 74104, Valencia, CA) according to the manufacturer's instructions. The purity and quantification for all RNA samples were done using the NanoVue spectrophotometer and the RNA quality was assessed on the Agilent BioAnalyzer 2100 (Agilent, Santa Clara, CA). All samples that present RIN > 8.0 were stored at -80°C until used in hybridization experiments.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Microarray-Based Gene Expression Analysis - Quick Amp Labeling (Agilent) according to the manufacturer's instructions, followed by RNAeasy 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 (G4112F) 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 1x44k array slides.
|
| Description |
Gene expression from Caco-2 after 3 hrs of interaction with STEC strain EC472, replicate 1
Caco-EC472#35
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software version 9.5.3.1 (Agilent) using default parameters (protocol GE1-v5_95 and Grid: 014850_D__F_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities but not included the normalization process. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
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| Submission date |
Apr 11, 2013 |
| Last update date |
Jan 06, 2018 |
| 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 |
GPL6480 |
| Series (1) |
| GSE45979 |
A HUS-associated STEC expressing a dicA transcriptional module is related to gene network dysregulation in Caco-2 |
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