|
| Status |
Public on Mar 19, 2013 |
| Title |
E. faecalis OG1RF exposed to 10μg/mL vancomycin for 60 minutes, replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
10μg/mL vancomycin, 60 minute exposure RNA
|
| Organism |
Enterococcus faecalis OG1RF |
| Characteristics |
growth phase: OD600 = 0.3
treatment: 10μg/mL vancomycin
time: 60 min
|
| Treatment protocol |
Upon reaching OD600 = 0.3, cells were treated with 10μg/mL vancomycin for 60 minutes
|
| Growth protocol |
Cells were grown in FMC medium supplemented with 10 mM glucose to OD600 of 0.3 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from homogenized E. faecalis cells grown in BHI medium to OD600 of 0.3 with or without exposure to antibiotics by repeated hot acid-phenol: chloroform extractions, and the nucleic acid was precipitated with 1 volume ice-cold isopropanol and one-tenth volume 3M sodium acetate (pH 5) at –20°C overnight. RNA pellets were resuspended in 80 mL 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 mL 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. Final RNA concentrations were determined in triplicate.
|
| 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 |
treatment: reference
|
| Growth protocol |
Cells were grown in Brain-Heart medium to OD600 of 0.5 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from homogenized E. faecalis cells grown in BHI medium to OD600 of 0.3 with or without exposure to antibiotics by repeated hot acid-phenol: chloroform extractions, and the nucleic acid was precipitated with 1 volume ice-cold isopropanol and one-tenth volume 3M sodium acetate (pH 5) at –20°C overnight. RNA pellets were resuspended in 80 mL 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 mL 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. Final RNA concentrations were determined in triplicate.
|
| 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 and scanned using a GenePix scanner (Axon Instruments).
|
| Scan protocol |
Hybridized slides were washed and scanned using a GenePix scanner (Axon Instruments, Inc., Union City, CA).
|
| 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 |
Mar 19, 2013 |
| Last update date |
Mar 19, 2013 |
| 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 (2) |
| GSE45300 |
Enterococcus faecalis OG1RF control vs. treatment with vancomycin |
| GSE45306 |
Enterococcus faecalis OG1RF control vs. treatment with ampicillin, bacitracin, cephalothin, or vancomycin |
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