|
| Status |
Public on Dec 25, 2007 |
| Title |
S. aureus vraSR mutant Daptomycin Treatment |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Daptomycin treated vraSR mutant KVR
|
| Organism |
Staphylococcus aureus |
| Characteristics |
vraSR- mutant of S. aureus N315
|
| Biomaterial provider |
Dr. Hiramatsu, Department of Bacteriology, Juntendo University, Tokyo, Japan
|
| Treatment protocol |
Overnight grown S. aureus KVR was inoculated in MHBc medium (20 ml) in a 50 ml Erlenmeyer flask and incubated at 37°C, with shaking at 200 rpm. Growth was measured at regular intervals at 600 nm until OD= ~0.4. Based on the GIC study daptomycin 2 µg ml-1 was added for 15 min of challenge.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total bacterial RNA was extracted from 3 ml of culture which was mixed with 6 ml of bacterial RNA protect solution (Qiagen, Valencia, CA) and the mixture was centrifuged at 3000 rpm for 20 min in swinging-bucket rotor centrifuge to collect the cells. Pellets were resuspended in 1 ml of Trizol (Invitrogen, Grand Island, NY) and the cells were broken using the FastPrep system (Qbiogene, Irvine, CA) at a speed of 6.0 for 40 seconds. From the broken cell lysate RNA was extracted as per the manufacturer’s instructions. Extracted RNA was purified using the RNeasy mini kit (Qiagen). DNase treated and purified mRNA was used for microarray analysis.
|
| Label |
Cy3 or Cy5
|
| Label protocol |
cDNA was generated by using random hexamers (Invitrogen) as primers for reverse transcription. The primers were annealed (70°C for 10 min, followed by snap-freezing in ice for 1 min) to total RNA (2 μg) and were extended with SuperScript II reverse transcriptase (Invitrogen) with 0.1 M dithiothreitol 12.5 mM dNTP/ aa-UTP (Ambion, Austin, TX) mix at 42°C overnight. Residual RNA was removed by alkaline treatment followed by neutralization, and cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified aminoallyl-modified cDNA was then labeled with Cy3 or Cy5 mono-functional NHS ester cyanogen dyes (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instruction.
|
| |
|
| Channel 2 |
| Source name |
Daptomycin treated S. aureus N315
|
| Organism |
Staphylococcus aureus |
| Characteristics |
Staphylococcus aureus N315
|
| Treatment protocol |
Overnight grown S. aureus N315 was inoculated in MHBc medium (20 ml) in a 50 ml Erlenmeyer flask and incubated at 37°C, with shaking at 200 rpm. Growth was measured at regular intervals at 600 nm until OD= ~0.4. Based on the GIC study daptomycin 10 µg ml-1 was added for 15 min of challenge.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total bacterial RNA was extracted from 3 ml of culture which was mixed with 6 ml of bacterial RNA protect solution (Qiagen, Valencia, CA) and the mixture was centrifuged at 3000 rpm for 20 min in swinging-bucket rotor centrifuge to collect the cells. Pellets were resuspended in 1 ml of Trizol (Invitrogen, Grand Island, NY) and the cells were broken using the FastPrep system (Qbiogene, Irvine, CA) at a speed of 6.0 for 40 seconds. From the broken cell lysate RNA was extracted as per the manufacturer’s instructions. Extracted RNA was purified using the RNeasy mini kit (Qiagen). DNase treated and purified mRNA was used for microarray analysis.
|
| Label |
Cy5 or Cy3
|
| Label protocol |
cDNA was generated by using random hexamers (Invitrogen) as primers for reverse transcription. The primers were annealed (70°C for 10 min, followed by snap-freezing in ice for 1 min) to total RNA (2 μg) and were extended with SuperScript II reverse transcriptase (Invitrogen) with 0.1 M dithiothreitol 12.5 mM dNTP/ aa-UTP (Ambion, Austin, TX) mix at 42°C overnight. Residual RNA was removed by alkaline treatment followed by neutralization, and cDNA was purified with a QIAquick PCR purification kit (Qiagen). Purified aminoallyl-modified cDNA was then labeled with Cy3 or Cy5 mono-functional NHS ester cyanogen dyes (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instruction.
|
| |
|
| |
| Hybridization protocol |
Purified labelled cDNA was hybridised with S. aureus genome microarray version 1.0 as previously described by Mongodin et al. (2003) and Riordon et al. (2007). Briefly, the slides were prehybridized in a buffer containing 5X SSC, 0.1% SDS and 0.1% BSA, and then washed and blown to dry. The two differentially labelled cDNA samples were dried and the resuspended in hybridization solution [40% deionized formamide, 5X SSC, 0.1% SDS, 0.6 µg/µl sheared Salmon sperm DNA (Ambion)]. The labelled cDNA samples were hybridized with the slides at 42 °C for 18-20 h. After hybridization, the slides were washed in low stringency buffer (2X SSC, 0.1% SDS) for 2 min, then in medium stringency (0.1x SSC, 0.1% SDS) buffer for 5 min and finally high stringency buffer (0.1x SSC) for 10 min.
|
| Scan protocol |
Hybridization signals were scanned using an Axon4000B scanner (Molecular Devices, Sunnyvale, CA ) with Acuity 4.0 software and scans were saved as TIFF image.
|
| Description |
Genes analyzed using these programs were further sorted and grouped based on their function using our in-house software Staphylococcus aureus Gene Sorter (SAGS). Several controls were employed to minimize the technical and biological variations and to ensure that the data obtained were of good quality. First, each ORF was present in triplicate on the array. Second, each RNA preparation was used to make probes for at least two separate arrays for which the incorporated dye was reversed.
|
| Data processing |
Scans were analyzed using TIGR-Spotfinder (www.tigr.org/software/) software and the local background was subsequently subtracted. The data set was normalized by applying the LOWESS algorithm using TIGR-MIDAS (www.tigr.org/software/) software. The normalized log2 ratio of test/reference signal for each spot was recorded. Genes with less than three data points were considered unreliable, and their data points were discarded. The averaged log2 ratio for each remaining gene on the six replicate slides was ultimately calculated. Significant changes of gene expression were identified with SAM (significance analysis of microarrays; www.tat.stanford.edu/~tibs/SAM/index.html software using one class mode.
|
| |
|
| Submission date |
Oct 31, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Arunachalam Muthaiyan, Brian Wilkinson |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
309-438-7244
|
| URL |
http://www.bio.ilstu.edu/Wilkinson/
|
| Organization name |
Illinois State University
|
| Department |
Biological Sciences
|
| Lab |
Microbiology
|
| Street address |
Julian Hall
|
| City |
Normal |
| State/province |
IL |
| ZIP/Postal code |
61790 |
| Country |
USA |
| |
|
| Platform ID |
GPL6059 |
| Series (1) |
| GSE9494 |
Transcriptional Profiling of Daptomycin Induced Staphylococcus aureus |
|
