|
| Status |
Public on Mar 30, 2016 |
| Title |
Y. pestis/Ciprofloxacin 0.016 µg / 120 min. replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
control
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: kimberley53
treated with: none (untreated control)
|
| Treatment protocol |
Ciprofloxacin exposure: The adjusted culture was divided into five samples of 400 ml each. Ciprofloxacin stock solutions (4 ml of 100X) were added to four culture samples to obtain final concentrations of 0.001, 0.016, 0.5 and 4 µg/ml, and the non-treated culture was used as growth control. Each of the five cultures was then further divided into 4×100 ml in 0.5 L flasks. The 0.5 L Erlenmeyer flasks were incubated at 28ºC, 150 rpm for 120 min. 40 ml of each culture were centrifuged at 3,200× g for 15 min at 4°C. Bacterial pellets were frozen by liquid nitrogen and stored at -70°C until RNA extraction.
|
| Growth protocol |
BHIA-plated Y. pestis Kimberley53 (5x10^5 CFU/ml) was suspended in MH broth (MHB) and incubated at 28oC, 150 rpm for 2 h for the adjustment of the culture to the MHB medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Residual DNA was removed by 15 min on-column digestion using the RNase-free DNase kit (QIAGEN).
|
| Label |
Cy3
|
| Label protocol |
Complementary RNA (cRNA) was produced from the total RNA samples and fluorescently labeled using the MessageAmpTM II-Bacteria kit (Ambion) according to the manufacturer’s instructions. Each experiment comprised of samples representing 4 different ciprofloxacin concentrations labeled with either Cy3-CTP or Cy5-CTP, which were co-hybridized with the growth control sample labeled with the 2nd dye (Cy5-CTP or Cy3-CTP, accordingly).
|
| |
|
| Channel 2 |
| Source name |
Ciprofloxacin 0.016 µg for 120 min
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: kimberley53
treated with: Ciprofloxacin 0.016 µg for 120 min
|
| Treatment protocol |
Ciprofloxacin exposure: The adjusted culture was divided into five samples of 400 ml each. Ciprofloxacin stock solutions (4 ml of 100X) were added to four culture samples to obtain final concentrations of 0.001, 0.016, 0.5 and 4 µg/ml, and the non-treated culture was used as growth control. Each of the five cultures was then further divided into 4×100 ml in 0.5 L flasks. The 0.5 L Erlenmeyer flasks were incubated at 28ºC, 150 rpm for 120 min. 40 ml of each culture were centrifuged at 3,200× g for 15 min at 4°C. Bacterial pellets were frozen by liquid nitrogen and stored at -70°C until RNA extraction.
|
| Growth protocol |
BHIA-plated Y. pestis Kimberley53 (5x10^5 CFU/ml) was suspended in MH broth (MHB) and incubated at 28oC, 150 rpm for 2 h for the adjustment of the culture to the MHB medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted using RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. Residual DNA was removed by 15 min on-column digestion using the RNase-free DNase kit (QIAGEN).
|
| Label |
Cy5
|
| Label protocol |
Complementary RNA (cRNA) was produced from the total RNA samples and fluorescently labeled using the MessageAmpTM II-Bacteria kit (Ambion) according to the manufacturer’s instructions. Each experiment comprised of samples representing 4 different ciprofloxacin concentrations labeled with either Cy3-CTP or Cy5-CTP, which were co-hybridized with the growth control sample labeled with the 2nd dye (Cy5-CTP or Cy3-CTP, accordingly).
|
| |
|
| |
| Hybridization protocol |
Each slide (containing 8 arrays) was hybridized with samples originating from 2 independent biological experiments representing the same time point, performed with Cy3-Cy5 dye swap. Each array represent hybridization of treated sample with its growth-control sample. Hybridization was done using the Agilent Gene Expression Hybridization Kit according to Agilent protocol for two-color Microarray-Based Gene Expression Analysis.
|
| Scan protocol |
Scanned on an Agilent DNA microarray G2505B scanner.
Images were quantified using Agilent Feature Extraction (FE) Software (version 9.5.1.1), with linear and LOWESS normalization.
|
| Description |
T2_120min_r1
|
| Data processing |
Agilent FE Software (9.5.1.1) was used for background subtraction and LOWESS normalization. Statistical analysis was performed using the Limma (Linear Models for Microarray Data) package from the Bioconductor project (http://www.bioconductor.org). The processed signal from the FE was read into Limma using the “read.maimages” function. Background subtraction and LOWESS normalization were performed for each array. Quantile normalization was applied between arrays. The Benjamini-Hochberg false discovery rate (FDR) was used to correct for multiple comparisons.
Values are the average of 2 independent biological experiments, normalized log2 ratio of ciprofloxacin exposed/non treated sample (Cy5/Cy3 of one biological experiment and Cy3/Cy5 of the duplicate biological experiment) .
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| |
|
| Submission date |
Mar 29, 2016 |
| Last update date |
Mar 30, 2016 |
| Contact name |
Ida Steinberger-Levy |
| E-mail(s) |
idas@iibr.gov.il
|
| Organization name |
IIBR
|
| Street address |
P.O. box 19
|
| City |
Ness-Ziona |
| ZIP/Postal code |
74100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL21664 |
| Series (1) |
| GSE79687 |
Y. pestis exposed to various concentrations of ciprofloxacin for various time points (treated/nontreated analysis in each array). |
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