|
| Status |
Public on May 07, 2010 |
| Title |
Stationary phase_biological replicate_3_technical replicate_2 |
| Sample type |
RNA |
| |
|
| Source name |
Moraxella catarrhalis RH4, BHI growth, stationary phase
|
| Organism |
Moraxella catarrhalis RH4 |
| Characteristics |
genotype: wild-type
genbank accession: CP002005
|
| Treatment protocol |
Bacteria treated with 2 volumes of RNAprotect bacteria reagent (Qiagen).
|
| Growth protocol |
Moraxella catarrhalis RH4 was grown overnight on brain heart infusion (BHI) agar plates at 37°C in the presence of 5% CO2. BHI broth cultures were grown at 37°C and 200 rpm till lag phase (OD620 = 0.2-0.3), exponential phase (OD620 = 1.2-1.4), and stationary phase (OD620 = 2.0-2.2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen) and DNA was removed by DNase digestion with the DNAfree kit (Ambion).
|
| Label |
5'Cy3-labeled random nonamers
|
| Label protocol |
Total RNA was labeled essentially as described by Ouellet et al. 2009 BMC Biotechnology (available from http://www.biomedcentral.com/1472-6750/9/97). Briefly, 10 µg of RNA was incubated for three hours with 7 µg of 5'Cy3-labeled random nonamers (TriLink Biotechnologies) and Superscript III reverse transcriptase (800 U, Invitrogen) in appropriate reaction conditions (1x First-Strand buffer, 5 mM DTT, 0.33 mM dNTPs, 21 mM actinomycin D (Sigma Aldrich), and 40 U RNaseOut). After first strand synthesis, RNA was degraded by incubating with sodium hydroxide, followed by reaction neutralization with hydrochloric acid. Labeled cDNA was purified and concentrated with CyScribe columns (GE Healthcare Life Sciences) followed by Micron-30 columns (Millipore).
|
| |
|
| Hybridization protocol |
2 µg of labeled cDNA was applied in duplicate to 4x72K custom design NimbleGen arrays. Overnight hybridization at 42°C and subsequent washing of arrays was performed according to the manufacturer’s instructions (available from www.nimblegen.com).
|
| Scan protocol |
Array images were acquired with a NimbleGen MS200 scanner following their standard operation protocol, using the autogain function per subarray.
|
| Description |
This sample is of stationary phase wild-type Moraxella catarrhalis RH4 grown in BHI medium. This is the third of three biological replicates used in this experiment and it is the second technical replicate
|
| Data processing |
The raw data (.pair) images were processed with NimbleScan software using the RMA algorithm. Background correction was used as implemented in the NimbleScan software package (Roche NimbleGen, Inc.)
|
| |
|
| Submission date |
May 03, 2010 |
| Last update date |
May 06, 2011 |
| Contact name |
Hester Bootsma |
| E-mail(s) |
h.bootsma@cukz.umcn.nl
|
| Phone |
+31-243666332
|
| Organization name |
Radboud University Medical Centre
|
| Department |
Pediatrics
|
| Lab |
Laboratory of Pediatric Infectious Diseases
|
| Street address |
Kapittelweg 29
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 EN |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL10378 |
| Series (1) |
| GSE21632 |
Expression profiling of Moraxella catarrhalis RH4 during in vitro growth in brain heart infusion (BHI) medium |
|
