|
| Status |
Public on Sep 15, 2012 |
| Title |
Bacteria suspension heated to a core temperature of 71°C - Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control bacteria at 37°C (common reference). Total RNA isolated from 5 indepedent cultures (pooled after cDNA labeling).
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: strain K12 substrain MG1655
growth protocol: Controls grown to an optimal temperature of 37°C
growth phase: stationary phase (grown to an OD600nm of 0.9)
|
| Treatment protocol |
After reaching an OD600nm of 0.9, cell cultures were placed immediately in a shaking water bath along with another BHI flask carrying a type T thermocouple connected to a MultiPaq 21 datalogger (Datapaq Inc.) for temperature profile and the process lethality values monitoring in real time. Bacteria suspension were heated at 58°C to process lethality values of 2 and 3, at 60°C to a process lethality value of 3, or until a temperature of 71°C was reached. Just after heating, cell suspension was cooled in an iced water bath under constant agitation (150 rpm) until the temperature dropped back to no less than 37°C in order to avoid cold stress. Cell suspensions were then centrifuged and the remaining cell pellets were treated with RNA protect bacteria reagent (Qiagen Inc.) prior to freezing at -80°C.
|
| Growth protocol |
For each biological replicate, frozen aliquots (500μl) of E. coli K12 stock cultures were subcultured in 10 ml of Brain Heart Infusion (BHI) and incubated overnight at 37°C. Cells were then sub-cultured (1% v/v) into 50ml BHI broth and incubated at 37°C for 24h under constant agitation (150 rpm). The bacterial suspensions were then inoculated (1% v/v) in 200ml BHI broth and grown in the same conditions to stationary phase (OD600nm of 0.9).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cell pellets were lyzed with lysozyme (Sigma Aldrich) and proteinase K (Qiagen Inc.) at room temperature. Total RNA was isolated from cell lysates using RNeasy Midi Kit according to the manufacturer's instructions (Qiagen Inc.). All RNA samples were then treated with DNase I (Ambion, Life technologies) before a phenol/chloroform purification and an overnight precipitation at -20°C in absolute ethanol containing LiCl 8M. After centrifugation, RNA pellets were air-dried in a vacuum concentrator ( vacufuge plus; Eppendorf).
|
| Label |
alexa fluor 555
|
| Label protocol |
Indirect labelling of RNA templates was based on the initial protocol described by Seidel and modified by Schroeder, Snesrud and Yang for the TIGR standard operating procedure (see Hegde P et al, 2000, Biotechniques 29(3):548-562). An amount of 5μg of total RNA were reverse transcribed for 3h at 42°C using Superscript II enzyme and random hexamers in the presence of 0.5mM each dATP, dCTP, dGTP, 0.3mM dTTP (Invitrogen, Life technologies) and 0.2mM 5-(3-aminoallyl)-dUTP (Sigma Aldrich). After hydrolysis of the remaining RNAs and purification on Qiaquick columns (Qiaquick PCR purification kit; Qiagen Inc.), the amine-modified cDNA obtained from control and heated cells were coupled to the succinimidyl ester of Alexa fluor 555 and 647 reactive dyes, respectively (Molecular probes, Life technologies). Unincorporated dyes were removed by purification on Qiaquick PCR purification columns.
|
| |
|
| Channel 2 |
| Source name |
Bacteria at 71°C
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: strain K12 substrain MG1655
growth protocol: Treatment 4 (core temperature of 71°C)
growth phase: stationary phase (grown to an OD600nm of 0.9)
|
| Treatment protocol |
After reaching an OD600nm of 0.9, cell cultures were placed immediately in a shaking water bath along with another BHI flask carrying a type T thermocouple connected to a MultiPaq 21 datalogger (Datapaq Inc.) for temperature profile and the process lethality values monitoring in real time. Bacteria suspension were heated at 58°C to process lethality values of 2 and 3, at 60°C to a process lethality value of 3, or until a temperature of 71°C was reached. Just after heating, cell suspension was cooled in an iced water bath under constant agitation (150 rpm) until the temperature dropped back to no less than 37°C in order to avoid cold stress. Cell suspensions were then centrifuged and the remaining cell pellets were treated with RNA protect bacteria reagent (Qiagen Inc.) prior to freezing at -80°C.
|
| Growth protocol |
For each biological replicate, frozen aliquots (500μl) of E. coli K12 stock cultures were subcultured in 10 ml of Brain Heart Infusion (BHI) and incubated overnight at 37°C. Cells were then sub-cultured (1% v/v) into 50ml BHI broth and incubated at 37°C for 24h under constant agitation (150 rpm). The bacterial suspensions were then inoculated (1% v/v) in 200ml BHI broth and grown in the same conditions to stationary phase (OD600nm of 0.9).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cell pellets were lyzed with lysozyme (Sigma Aldrich) and proteinase K (Qiagen Inc.) at room temperature. Total RNA was isolated from cell lysates using RNeasy Midi Kit according to the manufacturer's instructions (Qiagen Inc.). All RNA samples were then treated with DNase I (Ambion, Life technologies) before a phenol/chloroform purification and an overnight precipitation at -20°C in absolute ethanol containing LiCl 8M. After centrifugation, RNA pellets were air-dried in a vacuum concentrator ( vacufuge plus; Eppendorf).
|
| Label |
alexa fluor 647
|
| Label protocol |
Indirect labelling of RNA templates was based on the initial protocol described by Seidel and modified by Schroeder, Snesrud and Yang for the TIGR standard operating procedure (see Hegde P et al, 2000, Biotechniques 29(3):548-562). An amount of 5μg of total RNA were reverse transcribed for 3h at 42°C using Superscript II enzyme and random hexamers in the presence of 0.5mM each dATP, dCTP, dGTP, 0.3mM dTTP (Invitrogen, Life technologies) and 0.2mM 5-(3-aminoallyl)-dUTP (Sigma Aldrich). After hydrolysis of the remaining RNAs and purification on Qiaquick columns (Qiaquick PCR purification kit; Qiagen Inc.), the amine-modified cDNA obtained from control and heated cells were coupled to the succinimidyl ester of Alexa fluor 555 and 647 reactive dyes, respectively (Molecular probes, Life technologies). Unincorporated dyes were removed by purification on Qiaquick PCR purification columns.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed onto Agilent Escherichia coli gene expression microarray 8x15K according to the two-color microarray-based procaryote analysis protocol (Agilent technologies). Alexa fluor labelled cDNA obtained from the 5 replicates of control cultures at 37°C were pooled and used as a common reference for all hybridization. An aliquot of 300ng of this pooled control cDNA and 300ng of labelled cDNA from one of the experimental group were mixed together with blocking agent in the hybridization buffer (Gene expression hybridization kit, Agilent technologies) and loaded onto one of the eight microarray areas available on the slide. The biological replicates of the different treatment were randomly distributed on the slides. The microarray slides were incubated in the hybridization chamber at 65°C for 17h under constant vertical rotation. After hybridization, the slides were removed from the chambers and washed sequentially with GE washer buffers 1 and 2 (Agilent technologies).
|
| Scan protocol |
Microarray slides were scanned on a TECAN PowerScannerTM (TECAN group Ltd., Switzerland) and features extraction were processed with the Array-Pro 6.3 ANALYZER software (Media Cybernetics Inc., Bethesda, MD, USA).
|
| Description |
Biological replicate 2 of 5. Bacteria at 71°C compared to the common reference prepared from control bacteria at 37°C
Sample name: Treatment 4 - rep2
|
| Data processing |
LOWESS normalized data obtained from log2 of processed Red signal (treated)/processed Green signal (control) are given in the data table below. All microarray data were analyzed using R software and Limma package (part of Bioconductor). All the probes encoding for positive and negative controls were not used and have been removed. Only raw data superior to background values and with homogeneous fluorescent signals were considered (H < 0.2). Red and green signal heterogeneity was estimated as followed: H = |(trimmed mean of raw intensity - median of raw intensity)|/|(0.5*(trimmed mean of raw intensity + median of raw intensity)). After LOWESS normalization, the differences between the four heat-treatments were tested by Analysis of Variance (ANOVA) followed by a priori tests (P-value < 0.0001 and P-value FDR after Benjamini Yekutieli adjustment < 0.01). Six contrasts (Contrast1:T1 vs T2, Contrast2:T1 vs T3, Contrast3: T1 vs T4, Contrast4: T2 vs T3, Contrast5: T2 vs T4, Contrast6: T3 vs T4) were performed. Normalized Log2 ratio between treatments and the results of the statistical analysis have been linked as a supplementary file on the Series record.
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| Submission date |
Sep 03, 2012 |
| Last update date |
Sep 15, 2012 |
| Contact name |
ANTHONY GUERNEC |
| Organization name |
UNIVERSITE LAVAL
|
| Department |
ANIMAL SCIENCE
|
| Lab |
MICROBIOLOGY
|
| Street address |
2425 RUE DE L'AGRICULTURE
|
| City |
QUEBEC |
| State/province |
QUEBEC |
| ZIP/Postal code |
G1V0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13360 |
| Series (1) |
| GSE40557 |
Transcriptional analysis of E. coli whole genome during heat inactivation processes related to industrial cooking |
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