|
| Status |
Public on Nov 01, 2015 |
| Title |
MtuR mutant_late exp_2 vs. S. uberis 0140J_late exp_3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
bacterial RNA, MtuR, late exponential growth phase
|
| Organism |
Streptococcus uberis |
| Characteristics |
background/strain: 0140J
genotype/variation: MtuR mutant
growth phase: late exponential
replicate: 2
|
| Treatment protocol |
Two volumes of RNAprotect Bacterial Reagent (Qiagen) were immediately added to each sample, and the samples were incubated for 5 min at room temp. Samples were centrifuged (5,000 × g, 10 min), the supernatant was discarded and the cell pellets were stored at -80°C until RNA extraction was performed.
|
| Growth protocol |
Aliquots (30-60ml) of either S. uberis 0140J or MtuR mutant cultures were grown in Todd Hewit Broth (THB) with the addition of 1mM manganese at 37°C. Cultures were harvested at OD550nm of 0.43 for early logarithmic or OD550nm of 0.78 for late logarithmic growth phases.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by resuspension of cells in 1ml of RLT buffer (Qiagen) containing 1% (v/v) β-mercaptoethanol (Sigma-Aldrich). Cells were mechanically disrupted in a Mini-beadbeater-8 (Biospec) using acid-washed glass beads (Sigma). Following disruption, glass beads and cell debris were removed by centrifugation (17,000 × g, 10 sec). Supernatants were removed and mixed with an equal volume of 70% ethanol. Total RNA was extracted using an RNeasy column (Qiagen) according to the manufacturer’s instructions, with RNA finally eluted in 50µl of nuclease-free water. Contaminating DNA was removed from samples by DNase treatment using the DNA-free™ DNase Treatment kit (Ambion), and the resulting samples were further purified by ethanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
Fluorescently labeled probes for microarray hybridization were generated from 500ng total RNA using the MessageAmp II-Bacteria RNA amplification kit (Ambion) according to the manufacturer’s instructions. A total of 5µg of the resulting amplified antisense RNA (aRNA) was purified using the RNeasy minElute clean up kit (Qiagen), and hybridisation was performed using the Gene expression hybridization kit (Agilent). Samples were labeled with either Cy3 or Cy5 NHS ester reactive dyes (GE healthcare) according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
bacterial RNA, 0140J, late exponential growth phase
|
| Organism |
Streptococcus uberis |
| Characteristics |
background/strain: 0140J
genotype/variation: wild-type
growth phase: late exponential
replicate: 3
|
| Treatment protocol |
Two volumes of RNAprotect Bacterial Reagent (Qiagen) were immediately added to each sample, and the samples were incubated for 5 min at room temp. Samples were centrifuged (5,000 × g, 10 min), the supernatant was discarded and the cell pellets were stored at -80°C until RNA extraction was performed.
|
| Growth protocol |
Aliquots (30-60ml) of either S. uberis 0140J or MtuR mutant cultures were grown in Todd Hewit Broth (THB) with the addition of 1mM manganese at 37°C. Cultures were harvested at OD550nm of 0.43 for early logarithmic or OD550nm of 0.78 for late logarithmic growth phases.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by resuspension of cells in 1ml of RLT buffer (Qiagen) containing 1% (v/v) β-mercaptoethanol (Sigma-Aldrich). Cells were mechanically disrupted in a Mini-beadbeater-8 (Biospec) using acid-washed glass beads (Sigma). Following disruption, glass beads and cell debris were removed by centrifugation (17,000 × g, 10 sec). Supernatants were removed and mixed with an equal volume of 70% ethanol. Total RNA was extracted using an RNeasy column (Qiagen) according to the manufacturer’s instructions, with RNA finally eluted in 50µl of nuclease-free water. Contaminating DNA was removed from samples by DNase treatment using the DNA-free™ DNase Treatment kit (Ambion), and the resulting samples were further purified by ethanol precipitation.
|
| Label |
Cy5
|
| Label protocol |
Fluorescently labeled probes for microarray hybridization were generated from 500ng total RNA using the MessageAmp II-Bacteria RNA amplification kit (Ambion) according to the manufacturer’s instructions. A total of 5µg of the resulting amplified antisense RNA (aRNA) was purified using the RNeasy minElute clean up kit (Qiagen), and hybridisation was performed using the Gene expression hybridization kit (Agilent). Samples were labeled with either Cy3 or Cy5 NHS ester reactive dyes (GE healthcare) according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Cy3- and Cy5-labelled probes were mixed together in the appropriate combinations, with the fragmented RNA and hybridization buffer applied to the microarray hybridization chamber (Agilent Technologies) and incubated at 65°C for 17 hours with 10 RPM rotation. Following hybridization, microarrays were washed using Gene Expression Wash Buffer (Agilent Technologies).
|
| Scan protocol |
Arrays were scanned on an Agilent C scanner at 5 um using the XDR function at 10%. The images were then feature extracted using Agilent Feature Extraction version 10.5.1.1 using the GE2_105_Jan09 extraction protocol.
|
| Description |
Array 1_4
|
| Data processing |
Data were loess and scale normalized and analyzed for differential expression using limma. Linear models were fitted to the data to compare the MtuR mutant with the wild-type 0140J strain at both early and late logarithmic growth phases. The resultant p-values were adjusted for the false discovery rate. Genes were considered to be differentially expressed if the difference between the two strains was greater than 4 fold by analysis of the duplicate probes for each gene, and the comparison of the data sets preferentially showed an adjusted P-value of <0.001.
|
| |
|
| Submission date |
Mar 21, 2012 |
| Last update date |
Nov 01, 2015 |
| Contact name |
Sharon Egan |
| E-mail(s) |
sharon.egan@nottingham.ac.uk
|
| Organization name |
University of Nottingham
|
| Department |
School of Veterinary Medicine and Sciences
|
| Street address |
Sutton Bonington Campus
|
| City |
Sutton Bonington |
| State/province |
Leicestershire |
| ZIP/Postal code |
LE125RD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL14778 |
| Series (1) |
| GSE36677 |
Gene expression-based analysis of Streptococcus uberis 0140J and the MtuR (sub0472) mutant |
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