|
| Status |
Public on Aug 28, 2016 |
| Title |
S. typhimurium fosmidomycin treated 3 |
| Sample type |
RNA |
| |
|
| Source name |
Cells in early stage logarithmic growth
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 |
| Characteristics |
treatment: fosmidomycin treated
|
| Treatment protocol |
At early logarithmic growth (OD600 = 0.4) cells were exposed to subinhibitory concentrations of fosmidomycin or kanamycin and culture was continued for 20 minutes. A control sample of cells that were not exposed to antibiotics was also grown. After 20 minutes, the cells were collected by centrifugation, decanted, washed and flash frozen in the presence of an RNA preservative.
|
| Growth protocol |
S. typhimurium cells were inoculated from a confluent culture and grown at 37 C with shaking (200 rpm) in Luria-Bertani medium and monitored by optical density.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from all treatments was isolated using the Qiagen Rneasy RNA isolation kit according to the manufacturers protocol.
|
| Label |
Alexa Fluor 555
|
| Label protocol |
Ten micrograms of total RNA was converted to enriched mRNA using Applied Biosystem's (Ambion) MicrobExpressTm Kit using the manufacturers protocol. Enriched mRNA (200 ng) was converted to cRNA using Applied Biosystem's (Ambion) MessageAmp II-Bacteria RNA Amplification Kit. Amino-allyl-UTP was incorporated into the cRNA during the IVT reaction. The manufacturer's protocol was followed. AlexaFluor 555 (Invitrogen) was coupled to cRNA following the manufacturer's instructions and unincorporated dye was removed using RNeasy Mini Columns (Qiagen). cRNA coupled to fluorescent dye was fragmented using heat and zinc (2+) cations. The expected mean fragment length is 100-125 nucleotides. Fragmented cRNA is considered sufficent for use on MYcroarray custom arrays if the mean fragment length is less than 200 nucleotides.
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| |
|
| Hybridization protocol |
cRNA derived from all samples was coupled to Alexa Fluor 555. Ten micrograms of each target was hybridized separately to one array. Hybridization was performed for 19hrs at 50oC in a proprietary hybridization buffer. The arrays were washed as follows. The wash stock solution was 20X SSPE (3M NaCl, 20mM EDTA, 118.2mM NaH2PO4 and 81.8mM Na2HPO4). Each array was removed from the hybridization cassette in while submerged in 6X SSPE (22C), transferred to fresh 6X SSPE (22C) and stored briefly while the rest of the arrays were liberated from their cassettes (estimated storage time was <2min). Arrays were washed in batches of 5. Arrays were washed in 1X SSPE at 22C for 3 min (2 rounds) followed by 1X SSPE at 50C for 3 min, 1X SSPE at 22 C for 3 min and finally in 0.5 X SSPE at 22 C for 30 seconds. All washes were performed in baths while the wash solution was gently circulated by stir bar. Following the final wash, arrays were spun dry in a microarray centrifuge.
|
| Scan protocol |
The arrays were scanned in an Axon 4000B Scanner (Molecular Devices) set at 5μm per pixel resolution and 100% laser power. The PMT setting was adjusted to appreciate the maximum dynamic range of signal (0 to 65,000). This was accomplished by increasing the PMT (gain) until a few spots had some saturated pixels. The average number of spots with greater than 10% saturated pixels was 19.2 (out of a possible 26,346 SE spots).
|
| Description |
Fos_003
|
| Data processing |
Data was extracted from the scanned images using GenePix Pro Software (version 6.1.0.4). The gal file (“Load Array List” in GenePix) grid was positioned over the array image. For signal extraction, circular feature indicators (30m diameter) were centered over each spot. Median feature pixel intensity was extracted. Due to the manufacturing process, the area immediately surrounding a spot is not used to estimate background levels. MYcroarray INC states that when probes do not bind target, the residual signal is almost always lower compared with the surrounding area. To use local background values would over-estimate the contribution of background signal in the extracted signal intensity of each probe. Therefore, a global background correction algorithm within each array was used instead of a local background correction factor. The background value was determined by the 5th percentile darkest spot from the distribution of median signal intensities for all SE spots for each array (i.e., control spots and empty spots were ignored). Background corrected signal = Max (signal – background, 0.1). In other words, if background subtraction produces a value less than zero, 0.1 was used instead (i.e., the background corrected signal distribution is truncated slightly above zero to avoid having a zero or negative signal value). To adjust for differences in dye behavior, a scale factor was created to equalize signal across all arrays. Only probes that met the following criteria were used to calculate the scale factor: only SE spots were used; spots must have less than 10% saturated pixels; median signal had to be more than 3 fold local background; and all examined arrays had to have the spot qualify as a normalization spot. There were 11,789 qualifying spots.
Absent call (0=absent; 1=present)
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| |
|
| Submission date |
Aug 30, 2013 |
| Last update date |
Aug 28, 2016 |
| Contact name |
Andrew Koppisch |
| E-mail(s) |
andy.koppisch@nau.edu
|
| Organization name |
Northern Arizona University
|
| Department |
Chemistry
|
| Street address |
PO Box 5698
|
| City |
Flagstaff |
| State/province |
AZ |
| ZIP/Postal code |
86001 |
| Country |
USA |
| |
|
| Platform ID |
GPL17664 |
| Series (1) |
| GSE50480 |
Gene expression profiling of Salmonella enterica serovar Typhimurium exposed to subinhibitory concentrations of fosmidomycin and kanamycin |
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