|
| Status |
Public on Nov 19, 2015 |
| Title |
C. jejuni NCTC 11168 treated with (-)-α-Pinene, replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NA-4-untreated
|
| Organism |
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 |
| Characteristics |
treatment: none
|
| Treatment protocol |
62.5 ug/ml (-)-alpha pinene (dissolved in DMSO) was added to prepared culture (final concentration of DMSO was 0.048%). Culture was treated for 2 hours - shaking at 160 rpm, at 42 dC, microaerobically.
|
| Growth protocol |
Stock of Campylobacter jejuni NCTC 11168 was taken from stock stored on -80 dC and plated on Mueller Hinton agar. Culture was incubated at 42 dC for 24 hours, replated and incubated again at 42 dC for 24 hours, both times in microaerobic conditions with 5% O2. After that the culture was resuspended in MHB and shaken in microaerobic conditions at 42 dC until it reached OD600=0.2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy kit from Qiagen following manufacturer's instruction.
|
| Label |
Cy5
|
| Label protocol |
5 µg of total RNA were primed with 2 µl of 3mg/ml random hexamer primer at 70°C for 10 min, then reversed transcribed at 42°C overnight in the presence of 400 U SuperScript III RTase (Invitrogen) and 0.6 ul 25mM labeling mix (2:3 aa-dUTP to dTTP). The purified aminoallyl-labeled cDNA was then dissolved in 4.5 ul 0.1 M sodium carbonate buffer pH 9.3 and incorporated with 4.5ul Cy3 or Cy5 dye.
|
| |
|
| Channel 2 |
| Source name |
NA-2-treated
|
| Organism |
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 |
| Characteristics |
treatment: (-)-α-Pinene
|
| Treatment protocol |
62.5 ug/ml (-)-alpha pinene (dissolved in DMSO) was added to prepared culture (final concentration of DMSO was 0.048%). Culture was treated for 2 hours - shaking at 160 rpm, at 42 dC, microaerobically.
|
| Growth protocol |
Stock of Campylobacter jejuni NCTC 11168 was taken from stock stored on -80 dC and plated on Mueller Hinton agar. Culture was incubated at 42 dC for 24 hours, replated and incubated again at 42 dC for 24 hours, both times in microaerobic conditions with 5% O2. After that the culture was resuspended in MHB and shaken in microaerobic conditions at 42 dC until it reached OD600=0.2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy kit from Qiagen following manufacturer's instruction.
|
| Label |
Cy3
|
| Label protocol |
5 µg of total RNA were primed with 2 µl of 3mg/ml random hexamer primer at 70°C for 10 min, then reversed transcribed at 42°C overnight in the presence of 400 U SuperScript III RTase (Invitrogen) and 0.6 ul 25mM labeling mix (2:3 aa-dUTP to dTTP). The purified aminoallyl-labeled cDNA was then dissolved in 4.5 ul 0.1 M sodium carbonate buffer pH 9.3 and incorporated with 4.5ul Cy3 or Cy5 dye.
|
| |
|
| |
| Hybridization protocol |
The hybridization and wash were performed following (http://www.mycroarray.com/) standard protocol. Hybridization was for 24 hours at 42⁰C.
|
| Scan protocol |
Arrays were scanned at 532-nm (Cy3) and 635-nm (Cy5) wavelengths using GenePix 4100A (Molecular Devices, Sunnyvale, CA) following the manufacturer's protocol.
|
| Description |
C. jejuni NCTC 11168
Sample name: 965
Biological replicate 1
|
| Data processing |
Fluorescence intensities of each spot were extracted using GenePix Pro 7.0 (Molecular Devices). (i) After global background correction, the fluorescence intensity in each wavelength was log2 transformed and normalized using locally weighted linear regression (LOWESS) in statistical software R (http://www.r-project.org); (ii) for genes with more than one probe, only the genes with all the probes showing the same regulation trend were kept; (iii) T-test was performed to determine the differentially expressed genes.
|
| |
|
| Submission date |
Jul 29, 2014 |
| Last update date |
Nov 19, 2015 |
| Contact name |
Jasna Kovac |
| E-mail(s) |
jasna.kovac@bf.uni-lj.si
|
| Organization name |
University of Ljubljana, Biotechnical Faculty
|
| Department |
Department of Food Science and Technology
|
| Lab |
Food Microbiology Lab
|
| Street address |
Jamnikarjeva 101
|
| City |
Ljubljana |
| ZIP/Postal code |
1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL19011 |
| Series (1) |
| GSE59879 |
Adaptive mechanism of Campylobacter jejuni to treatment with a novel CmeABC efflux pump inhibitor (-)-α-Pinene |
|
