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| Status |
Public on Sep 27, 2013 |
| Title |
NrrF mutant strain_LJ001_Fe- replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Iron depleted growth conditions; cells harvested at 3 hrs
|
| Organism |
Neisseria gonorrhoeae |
| Characteristics |
strain: LJ001
genotype: NrrF_mutant
|
| Treatment protocol |
Fe-deplete (CDM-0) and Fe-replete medium (CDM-10, supplemented with 10 Fe(NO3)3) were prepared as previously reported. Jackson LA, Ducey TF, Day MW, Zaitshik JB, Orvis J, et al. (2010) Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon. J Bacteriol 192: 77-85
|
| Growth protocol |
All strains were routinely grown on GCB agar (Becton Dickinson) supplemented with 2% IsoVitale X (Becton Dickinson) from frozen stock cultures under 5% CO2 atmosphere at 37C. Cultures were grown in CDM-0 (Fe-deplete) to OD600 = 0.200 to Fe starve the cells. Half of the culture was transferred into another 500 ml flask and 62.5 ml of CDM-0 added to each flask to reach 125 ml and Fe(NO3)3 added to the CDM-10 flask for a 10uM final concentration. The cultures were then allowed to grow to stationary phase in a 37C shaking incubator (225rpm) with cells being collected at 3hr time point for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated as previously desribed, Jackson LA, Ducey TF, Day MW, Zaitshik JB, Orvis J, et al. (2010) Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon. J Bacteriol 192: 77-85, with the following changes: Total RNA was DNA digested [1U RQ1 RNase-Free DNase per 1g RNA (Promega)] followed by RNA clean-up according to TriReagent protocol (Molecular Research Center, Inc.). Quantitation was done using the NanoDrop ND-100 (NanoDrop Products) and RNA integrity assessed with Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer
|
| Label |
Biotin
|
| Label protocol |
cDNA was generated from 10g of DNase-treated total RNA using an Applied Biosystems High-Capacity cDNA Archive Kit. A. thaliana mRNA positive controls (SpotReport mRNA: Cab, RCA, and RBCL) were spiked into each reverse transcription reaction each at a final concentration of 1ng each (Stratagene). Post-synthesis labeling of cDNA was accomplished using the Mirus Label IT Array Biotin Labeling Kit. The cDNA was purified after the removal of RNA template pre-labeling and after biotin-labeling of the cDNA, using a QIAquick PCR Purification Kit (Qiagen). 3g of biotin-labeled cDNA was hybridized to the custom 12K N. gonorrhoeae expression array, according to the CombiMatrix hybridization protocol. Jackson LA, Dyer D (2012) Protocol for gene expression profiling using DNA microarrays in Neisseria gonorrhoeae. In: MacKenzie CR, editor. Diagnosis of Sexually Transmitted Diseases: Humana Press. pp. 343-357.
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| |
|
| Hybridization protocol |
Arrays were hybridized at 50C for 14 hours before electrochemical detection using an ElectraSense Reader (CombiMatrix).
|
| Scan protocol |
Arrays were scanned using ElectraSense Reader (CombiMatrix)
|
| Data processing |
Data files generated by the ElectraSense Reader software were imported into GeneSpringGX 12.5 for analysis after background subtraction of the “no oligo” control probes.
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| |
|
| Submission date |
Jul 03, 2013 |
| Last update date |
Sep 27, 2013 |
| Contact name |
Lydgia Jackson |
| E-mail(s) |
lydgia-jackson@ouhsc.edu
|
| Organization name |
OUHSC
|
| Department |
Micorbiology and Immunology
|
| Lab |
David Dyer
|
| Street address |
975 NE 10th Street
|
| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
| |
|
| Platform ID |
GPL17407 |
| Series (1) |
| GSE48526 |
Control of RNA stability by NrrF, an iron-regulated small RNA in Neisseria gonorrhoeae. |
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