|
| Status |
Public on Sep 19, 2013 |
| Title |
10min_RB2RT2_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MEMα + Ethanol (control) (10 min)
|
| Organism |
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 |
| Characteristics |
strain: NCTC11168
optical density 600nm: 0.2
treatment: Ethanol (control)
|
| Treatment protocol |
Benzylisothiocyanate (Sigma) stock solutions were prepared in absolute ethanol. Fresh dilutions were made in absolute ethanol. 500µL of diluted benzylisothiocyanate in ethanol was added to 25mL mid-log grown cells in dMEM culture media (treated sample), to achieve a final benzylisothiocyanate concentration of 2µg/mL in culture media. As a control, the same volume of absolute ethanol was added to 25mL cells mid-log grown cells in dMEM culture media. Total RNA extraction was carried out 10 min or 15 min after addition of diluted benzylisothiocyanate or ethanol.
|
| Growth protocol |
Overnight MH biphasic cultures were used to inoculate 25ml of MEMα (supplemented with 20mM pyruvate) to an OD600nm of 0.01. C. jejuni NCTC11168 cells were then grown to mid-log phase (OD600nm=0.2) at 37°C under microaerophilic conditions (10% O2, 5% CO2, 85% N2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 10 or 15 min of benzylisothiocyanate or ethanol treatment, 2.5mL cold RNA degradation stop solution (10% buffer-saturated phenol pH 4.3 in ethanol) were added to prevent RNA decay. Bacterial cells were harvested by centrifugation, resuspended in TE buffer followed by total RNA isolation using the hot phenol-chloroform method as described previously (Tao et al., 1999). Extracted RNA samples were treated twice with DNase-I (Epicenter) and purified using the RNeasy kit (Qiagen) followed by PCR amplification to confirm the absence of contaminating genomic DNA in the samples. The Experion RNA STDsens Analysis Kit (Bio-Rad Laboratories) was used to quantify and confirm RNA integrity of the extracted RNA samples. RNA samples were stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
Preparation of fluorescently labelled cDNA and microarray slide hybridization was performed as described previously (Palyada et al., 2004). Briefly, 16µg of C. jejuni (from benzylisothiocyanate treated samples or ethanol control samples) extracted RNA was reversed transcribed in presence of 10µg random hexamers, 0.5mM dGTP, dATP, dCTP, 0.16mM dTTP, and 0.34mM aminoallyl dUTP using SuperScript II (Invitrogen) for 2 hours at 42°C. The reaction was stopped by the addition of 50mM EDTA and 10 N NaOH and incubated at 65°C for 20 minutes. The reaction was neutralized by addition of 5M acetic acid and the aminoallyl-labelled cDNA was purified from non-incorporated aminoallyl dUTPs using Micron YM-30 spin columns (Millipore). The purified aminoallyl-labelled cDNA was coupled to Cy3 or Cy5 monoreactive fluors (Amersham) using the method described previously (Palyada et al., 2004). Following the coupling of fluorescent dyes to cDNA, the cDNA samples were purified using the PureLink PCR Purification Kit (Invitrogen) according to the manufacturer’s specifications.
|
| |
|
| Channel 2 |
| Source name |
MEMα + Benzylisothiocyanate 2µg/mL (10 min)
|
| Organism |
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 |
| Characteristics |
strain: NCTC11168
optical density 600nm: 0.2
treatment: 10 min Benzylisothiocyanate 2µg/mL
|
| Treatment protocol |
Benzylisothiocyanate (Sigma) stock solutions were prepared in absolute ethanol. Fresh dilutions were made in absolute ethanol. 500µL of diluted benzylisothiocyanate in ethanol was added to 25mL mid-log grown cells in dMEM culture media (treated sample), to achieve a final benzylisothiocyanate concentration of 2µg/mL in culture media. As a control, the same volume of absolute ethanol was added to 25mL cells mid-log grown cells in dMEM culture media. Total RNA extraction was carried out 10 min or 15 min after addition of diluted benzylisothiocyanate or ethanol.
|
| Growth protocol |
Overnight MH biphasic cultures were used to inoculate 25ml of MEMα (supplemented with 20mM pyruvate) to an OD600nm of 0.01. C. jejuni NCTC11168 cells were then grown to mid-log phase (OD600nm=0.2) at 37°C under microaerophilic conditions (10% O2, 5% CO2, 85% N2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 10 or 15 min of benzylisothiocyanate or ethanol treatment, 2.5mL cold RNA degradation stop solution (10% buffer-saturated phenol pH 4.3 in ethanol) were added to prevent RNA decay. Bacterial cells were harvested by centrifugation, resuspended in TE buffer followed by total RNA isolation using the hot phenol-chloroform method as described previously (Tao et al., 1999). Extracted RNA samples were treated twice with DNase-I (Epicenter) and purified using the RNeasy kit (Qiagen) followed by PCR amplification to confirm the absence of contaminating genomic DNA in the samples. The Experion RNA STDsens Analysis Kit (Bio-Rad Laboratories) was used to quantify and confirm RNA integrity of the extracted RNA samples. RNA samples were stored at -80°C.
|
| Label |
Cy5
|
| Label protocol |
Preparation of fluorescently labelled cDNA and microarray slide hybridization was performed as described previously (Palyada et al., 2004). Briefly, 16µg of C. jejuni (from benzylisothiocyanate treated samples or ethanol control samples) extracted RNA was reversed transcribed in presence of 10µg random hexamers, 0.5mM dGTP, dATP, dCTP, 0.16mM dTTP, and 0.34mM aminoallyl dUTP using SuperScript II (Invitrogen) for 2 hours at 42°C. The reaction was stopped by the addition of 50mM EDTA and 10 N NaOH and incubated at 65°C for 20 minutes. The reaction was neutralized by addition of 5M acetic acid and the aminoallyl-labelled cDNA was purified from non-incorporated aminoallyl dUTPs using Micron YM-30 spin columns (Millipore). The purified aminoallyl-labelled cDNA was coupled to Cy3 or Cy5 monoreactive fluors (Amersham) using the method described previously (Palyada et al., 2004). Following the coupling of fluorescent dyes to cDNA, the cDNA samples were purified using the PureLink PCR Purification Kit (Invitrogen) according to the manufacturer’s specifications.
|
| |
|
| |
| Hybridization protocol |
Fluorescently labelled cDNA samples from the benzylisothiocyanate-treated or ethanol control samples were combined, vacuum dried, and resuspended in 36µl of hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 25µg salmon sperm DNA). NCTC11168 microarray slides (Palyada et al., 2004; Stintzi, 2003) were prepared for hybridization by incubation in slide hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 1% BSA) for 45 minutes at 42°C. The fluorescently labelled, pooled cDNA samples were hybridized to the microarray slide a under glass coverslip which was placed in a humidified chamber (ArrayIt) overnight at 42°C protected from light. Following overnight incubation, the microarray slide was washed in 100mL of 2X SSC, 0.1% SDS solution for 5 min at 42°C. The slide was subsequently washed in 100mL of 0.1X SSC, 0.1% SDS at room temperature for 10 min and followed by an additional 4x 1 min washes in 0.1X SSC, 0.1% SDS solution. The slide was rinsed briefly in distilled H2O and dried by centrifugation.
|
| Scan protocol |
Microarray slides were scanned at 532 nm (Cy3) and 635 nm (Cy5) wavelengths using a laser-activated confocal scanner (ScanArray Gx, Perkin Elmer) at a 10µm resolution.
|
| Description |
10 minutes treatment Biological replicate 2 technical replicate 2 column 1
|
| Data processing |
Data collection was performed using the GenePix Pro 4.0 software (Axon Instruments) which determines the signal intensity for each spot on the microarray. Hybridization abnormalities or spots that were localized within regions of hybridization were removed from the analysis and the raw fluorescent intensities for each spot were subtracted from the background fluorescence of the microarray. Spots that had background-subtracted intensity values less than three times the standard deviation of the local background in both Cy3 and Cy5 channels were subsequently removed from data analysis. Next, MIDAS software (available from TIGR; http://www.tigr.org/software/) was used along with locally weighted linear regression (Lowess) to normalize the fluorescent intensity values for both Cy3 and Cy5 channels. The ratio of the fluorescent intensity values in the Cy3 channel to the Cy5 channel was calculated and converted into a log2 value.
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| |
|
| Submission date |
Apr 05, 2013 |
| Last update date |
Sep 19, 2013 |
| Contact name |
Virginie Dufour |
| E-mail(s) |
virginie.dufour.pro@gmail.com
|
| Organization name |
Université Rennes 1
|
| Street address |
263 avenue du General Leclerc
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL6293 |
| Series (1) |
| GSE45823 |
Campylobacter jejuni transcriptomic response to benzylisothiocyanate |
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