|
| Status |
Public on Dec 20, 2011 |
| Title |
E. faecalis relArelQ double mutant, 30 min mupirocin treatment, replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
relAQ mutant treated with mupirocin for 30 min RNA
|
| Organism |
Enterococcus faecalis OG1RF |
| Characteristics |
strain: OG1RF
genotype: relAQ mutant
growth phase: early-log mupirocin stressed
treatment: mupirocin
treatment duration: 30 min
|
| Treatment protocol |
Mupirocin treatment for 30 min.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate RNA from E. faecalis, cells were harvested by centrifugation at 4°C and then treated with the RNA protect reagent (QIAGEN, Inc., Chatsworth, CA). Total RNA was isolated from homogenized E. faecalis cells by the hot acid-phenol method as described previously (Abranches, 2006). RNA pellets were resuspended in nuclease-free H2O, and treated with DNase I (Ambion) at 37°C for 30 minutes. RNA concentrations were determined using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and 1 µL samples were run on an agarose gel to verify RNA integrity. The RNA was purified again using the RNeasy mini kit (Qiagen), including a second on-column DNase treatment that was performed as recommended by the supplier.
|
| Label |
Cy3
|
| Label protocol |
Once RNA was purified as described above, cDNA was generated from 2ug RNA per sample as described by the protocol available from the Pathogen Functional Genomics Research Center (PFGRC) using Invitrogen Superscript III Reverse Transcriptase (Invitrogen, Gaithersburg, MD) to increase cDNA yields. Purified cDNAs were coupled to Cy3 (experimental samples) or Cy5 (reference cDNA).
|
| |
|
| Channel 2 |
| Source name |
wild type mid-log RNA
|
| Organism |
Enterococcus faecalis OG1RF |
| Characteristics |
strain: OG1RF
genotype: wild-type
growth phase: mid-log
treatment: none
sample type: reference
|
| Growth protocol |
Cells were grown in Brain-Heart medium to OD600 of 0.5 in a 5% CO2 incubator at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate RNA from E. faecalis, cells were harvested by centrifugation at 4°C and then treated with the RNA protect reagent (QIAGEN, Inc., Chatsworth, CA). Total RNA was isolated from homogenized E. faecalis cells by the hot acid-phenol method as described previously (Abranches, 2006). RNA pellets were resuspended in nuclease-free H2O, and treated with DNase I (Ambion) at 37°C for 30 minutes. RNA concentrations were determined using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and 1 µL samples were run on an agarose gel to verify RNA integrity. The RNA was purified again using the RNeasy mini kit (Qiagen), including a second on-column DNase treatment that was performed as recommended by the supplier.
|
| Label |
Cy5
|
| Label protocol |
Once RNA was purified as described above, cDNA was generated from 2ug RNA per sample as described by the protocol available from the Pathogen Functional Genomics Research Center (PFGRC) using Invitrogen Superscript III Reverse Transcriptase (Invitrogen, Gaithersburg, MD) to increase cDNA yields. Purified cDNAs were coupled to Cy3 (experimental samples) or Cy5 (reference cDNA).
|
| |
|
| |
| Hybridization protocol |
The slides were hybridized to a mixture containing equal amounts of test and reference cDNA for 16 h at 42°C in a MAUI hybridization chamber (BioMicro Systems). Hybridized slides were washed.
|
| Scan protocol |
Slides were scanned using a GenePix scanner (Axon Instruments, Inc., Union City, CA).
|
| Description |
ΔrelAQ Mup30-3
|
| Data processing |
Data were analyzed using software available at the J. Craig Venter Institute PFGRC website. Single-channel images of the slides were loaded into Spotfinder and overlaid. A spot grid was created according to PFGRC specifications and manually adjusted to fit all spots. The Microarray Data Analysis System (MIDAS) software was used to normalize the spot intensity data using LOWESS and iterative log mean centering with default setting. MIDAS also utilized In-Slide Replicate Analysis to average the intensities of the spots that were replicated on the microarray slide. Spots that were missing or labeled as “bad” during the upstream processes in 50% of the slides were cut off in the output files, which also did not include control spots printed on the slides. Statistical analysis was carried out with BRB array tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) with a cutoff P value of 0.001.
|
| |
|
| Submission date |
Dec 19, 2011 |
| Last update date |
Dec 20, 2011 |
| Contact name |
Jessica K Kajfasz |
| E-mail(s) |
jkajfasz@dental.ufl.edu
|
| Organization name |
University of Florida
|
| Department |
Oral Biology
|
| Lab |
Lemos-Abranches
|
| Street address |
1395 Center Drive
|
| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
| |
|
| Platform ID |
GPL15039 |
| Series (1) |
| GSE34561 |
Enterococcus faecalis OG1RF vs. Δrel mutants under control or mupirocin-treated conditions |
|
