|
| Status |
Public on Sep 08, 2014 |
| Title |
Ecoli_NP-TiO2_5h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
E.coli, NP-TiO2 exposed culture, 5h in 10 mM NaCl 100mg/L NP-TiO2, replicate 1
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
incubation medium: 10 mM NaCl + 100mg/l TiO2 nanoparticles
incubation condition: In the dark, 5h, 20°C, 150 rpm
|
| Treatment protocol |
After 5 h of NP-TiO2 (or mQ water for the control) exposure, cells were incubated for 5 min with two volumes of RNAprotect Bacteria Reagent (Qiagen SAS, France) at room temperature. Cells were then pelleted by centrifugation (7,000 g, 10 min) and the pellet stored at -80°C.
|
| Growth protocol |
Cell exposure to NP-TiO2 was conducted in 50 ml of sterile Milli-Q water supplemented with 10 mM NaCl. Briefly, 500 µl of the E. coli bacterial suspension and 500 µl of the NP-TiO2 stock suspension (or mQ water for the control) were prepared as previously described and then added to the NaCl solution to obtain final concentrations of 10E7 cells/ml and 100 mg/l of TiO2 nanoparticles. The flasks were then incubated at 20°C on a dark rotary shaker (150 rpm) for 5 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells with the UltraClean Microbial RNA isolation kit (MO BIO, CA, USA). After extraction, contaminating DNA was digested with DNAse I (Sigma Aldrich) and total RNA was purified by phenol/chloroform extraction and ethanol precipitation. RNA purity and quantity was assess by optical density measurements (OD 260/280 and OD 260/230) and RNA integrity was checked with the Bioanalyseur 2100 (Agilent, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
Total RNA was reverse transcribed, amplified, and labeled with cyanine 3 (Cy3) using the low Input Quick Amp WT labeling kit (Agilent, CA, USA). The labeled cRNA (antisense) was then purified with the RNeasy mini spin columns (Qiagen). Quantification and measure of incorporation level of Cy3 were done with a NanoDrop ND-1000 (Thermo Scientific, DE, USA). The labeled cRNA was fragmented with Agilent fragmentation buffer 30 min at 60°C and RNA fragmentation was checked on BioAnalyseur.
|
| |
|
| Hybridization protocol |
For each samples, 825 ng of cRNA with a specific activity of at least 15 pmol of Cy3 per µg of cRNA (according to the manufacturer’s protocol) was hybridized in presence of GE Hybridization Buffer HI-RPM (Agilent) on the Agilent E. coli microarray (8x15K slide format, design ID: 020097) 17h at 65°C in a rotation oven (Agilent). Slides were washed successively 1 min in GE wash buffer 1 and 2 (Agilent) and dried by centrifugation.
|
| Scan protocol |
Microarray was scanned at 5 µm resolution with GenePix 4100 A (Molecular Devices, CA, USA).
|
| Description |
Expression profile of E.coli exposed to TiO2 nanoparticles, biological replicatee 1
|
| Data processing |
Image analysis was performed with GenePix Pro 7 software (Molecular Device), background was defined with the “Local features background median” method. Quantile normalization was applied on the total number of spots in R (version 2.11.1) with the LIMMA software package. Spots corresponding to the E. coli MG1655 K12 species were extracted.
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| |
|
| Submission date |
Mar 17, 2014 |
| Last update date |
Sep 08, 2014 |
| Contact name |
Bénédicte SOHM |
| E-mail(s) |
sohm5@univ-lorraine.fr
|
| Phone |
0033372748942
|
| Organization name |
CNRS
|
| Lab |
UMR 7360
|
| Street address |
8 rue du Gal Delestraint
|
| City |
METZ |
| ZIP/Postal code |
57070 |
| Country |
France |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE55972 |
Effect of Titanium dioxide nanoparticles on Escherichia coli K12 MG1655 genes expression |
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