|
| Status |
Public on Jun 15, 2015 |
| Title |
KCl 24 h_rep3 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Salmonella exposed to KCl for 24 h
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74 |
| Characteristics |
agent: KCl
time point: 24 h
strain: ST4/74
biological replicate: 3
|
| Growth protocol |
Salmonella Typhimurium 4/74 grown to early stationary phase in LB, statically at 24˚C, were harvested and resuspended in 10 ml of LO medium (10 g/l tryptone, 5 g/l yeast extract) containing 9.25% KCl and incubated statically at 24˚C for 24 h
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1)Cultures exposed to 9.25% KCl (10 ml) for 24 h were harvested by centrifugation. 2) The cell pellet was dissolved in 1 ml TRIzol on ice (Invitrogen, Cat: 15596026). 3) The Trizol/RNA solution was transferred to Phase-Lock-Tubes (5Prime, Cat: 2302830), 400 µl CHCl3 was added and tube inverted for 6-8 times; samples were then incubated for 2-5 min at room temperature. 2) Samples were centrifuged for 15 min at 15°C at full speed and the aqueous phase transferred to a new 1.5 ml microfuge tube. 3) Isopropanol (450 µl) was added to the sample and immediately mixed by inverting and incubated at-20°C overnight. 4) Samples were centrifuged for 30 min at full speed, the supernatant discarded and the pellet washed with 350µl 70 % EtOH, and centrifuged for 10 min (4°C) full speed. The supernatant was discarded and pellet air-dired. 5) The RNA pellet was re-suspended in 80 µl RNase free water, heated to 65˚C for 10 min at 850 rpm in a shaking heat block and nucleotide concentration measured on Nanodrop. 6) Samples were treated with DNase I (Fermentas, EN0521) and Supease-In RNAse Inhibitor (1 µl; Ambion, AM2694). Samples were adjusted to a concentration > 1,300 ng/l with RNase free water. 7) A NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, Delaware, USA) was used to quantify RNA concentrations. The Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) was used to assess RNA quality according to manufacturer’s instructions
|
| Label |
Cy3
|
| Label protocol |
1)Random priming reactions were set up in 1.5 ml microfuge tubes. Total RNA (10 µg) and 5 µg of random hexamers (Invitrogen, Cat: 48190011) were added to a final volume of 9.4 µl in RNase free water. 2) Samples incubated at 70 ˚C for 5 min and chilled on ice for 10 min. 3) RT reaction mix prepared by using the Stratagene AffinityScript multi-temperature Reverse Transcriptase by adding the following to each tube: 2 µl of 10 X RT buffer; 2 µl of 0.1 M DDT; 0.6 µl of 50 X dNTP’s (25 mM dATP, dGTP, dTTP and 10 mM dCTP[GE Healthcare Lifesciences, Cat: 27-2035-02]); 2 µl of Cy3 dCTP (1 mM stock, GE Healthcare Lifesciences, Cat: PA55321); 4 µl of reverse transcriptase. 4) Samples were vortexed to mix and incubated shaking at 25 ˚C for 10 min. 5) Samples then incubated overnight at 42 ˚C. 6) Freshly prepared 0.1 M NaOH (15 µl) was added and the RNA hydrolysed at 70 ˚C for 10 min. Freshly prepared 0.1 M HCl (15 µl) was added to neutralise the alkali. Samples were placed on ice to cool to room temperature. 7) Qia-quick PCR purification kit (Qiagen, Cat: 28104) was used to remove unincorporated/quenched cy dyes. Samples eluted into 40 µl RNase free water and dried-down on speed vac to 18 µl.
|
| |
|
| Channel 2 |
| Source name |
Reference genomic DNA
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74 |
| Characteristics |
strain: ST4/74
reference: stationary phase at 37˚C
|
| Growth protocol |
Salmonella enterica serovar Typhimurium 4/74 grown to stationary phase at 37˚C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA isolated using the Qiagen 'Genomic DNA' Kit (Cat. No.: 19060 for the buffer kit; 10243 for the columns).
|
| Label |
Cy5
|
| Label protocol |
1) Chromosomal DNA (2 µg) added to sterile microfuge tube and brought to 21 µl with RNase free water (Sigma, W4502-1L). 2) 20 µl each of 2.5 X Random primer/reaction buffer mix from the Invitrogen BioPrime® DNA Labelling System (18094011) was added to sample and boiled for 5 min, then put on ice for 5 min. 3) On ice, 5 µl of 10X dNTP mix (10X dNTP mix: 1.2 mM each dATP, dGTP, dTTP; 0.6 mM dCTP; 10 mM Tris pH 8.0; 1 mM EDTA;); 3 µl of Cy5 dCTP (1 mM stock, GE Helathcare Lifesciences, Cat: PA55321); and 1 µl Klenow enzyme from the kit were added. 4) Samples were spun briefly and the reaction mix incubated at 37 ˚C overnight. 5) A Qia-quick PCR purification kit (Qiagen Cat: 28104) was used to remove unincorporated/quenched Cy dyes. Samples were eluted into 80 µl of RNase free water. This protocol supplies sufficient labelled DNA for 9 hybridisations. Samples adjusted to a volume of 50 µl using speed vac.
|
| |
|
| |
| Hybridization protocol |
1)Prepared Cy 5 labelled gDNA (6 µl) was added to Cy3 labelled cDNA (18 µl) for a total volume of 24 µl. 2) To each sample, 6µl of blocking solution (Agilent 5188-5973) and 30 µl of :hybridisation buffer (Agilent 5188-6420) were added. 3) Samples were boiled for 2 min at 95oC and centrifuged for 1 min at 13000 rpm. 4) The gasket slide was placed in hybridization chamber rubber and 50 µl of sample placed directly into centre of gasket slide. 5) When all samples were dispensed the array slide was inserted into the hybridisation chamber, sealed and placed in pre-heated hybridisation over (65˚C) at 10 rpm overnight. 6) Slides were removed from chamber washed according to manufacturer’s instructions (http://www.chem.agilent.com/Library/usermanuals/Public/G2534-90004_HybridizationChamber_User.pdf). 7) Array slides were cleaned with inert gas before measurement.
|
| Scan protocol |
Scans were carried out using an Agilent DNA Microarray Scanner, Model G2565, at at 5 µm resolution with Green and Red PMT values set to 100 % and an XDR value of 0.1. Images generated were saved as multi-image .tiff files.
|
| Description |
KCl_test_24h_b3
|
| Data processing |
Agilent Feature extraction software was used to process the scanned images of the arrays. The data generated was then analysed using GeneSpring version 7.3 (Agilent Technologies, Santa Clara, California) based on the log ratio of median Cy3/Cy5 signal normalised to the 50th percentile.
|
| |
|
| Submission date |
Jun 14, 2015 |
| Last update date |
Jun 15, 2015 |
| Contact name |
Sarah Finn |
| E-mail(s) |
[email protected]
|
| Organization name |
Centre for Food Safety
|
| Street address |
University College Dublin
|
| City |
Dublin 4 |
| ZIP/Postal code |
4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL17132 |
| Series (1) |
| GSE69857 |
Exposure of Salmonella enterica serovar Typhimurium to three humectants used in the food industry: NaCl, KCl and glycerol |
|
