|
| Status |
Public on Apr 12, 2009 |
| Title |
Saccharopolyspora rif6 RNA at 72h biological rep.B |
| Sample type |
RNA |
| |
|
| Source name |
RNA extracted from Saccharopolyspora erythraea rif6 mutant after 72h of growth
|
| Organism |
Saccharopolyspora erythraea |
| Characteristics |
strain: NRRL 2338 rif6
|
| Growth protocol |
For shake-flask experiments, spores in frozen aliquots were collected by centrifugation, re-suspended in medium 707 (for rehydration), and readily separated by vortexing. Individual aliquots (about 5 x 108 spores) were used to inoculate each 500 ml buffled Erlenmeyer flask containing 50 ml liquid media above described. Cultures were incubated at 30°C with shaking at 250 rpm in R3 medium. R3 composition: 5 g yeast extract, 0.1 g casamino acids, 3 g L-proline, 10 g MgCl2 • 6H2O, 4 g CaCl2 • 2H2O, 0.2 g K2SO4, 0.05 g KH2PO4, 5.6 g TES, 10 g glucose, PH 7.2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each time point, RNA was extracted from mycelium pellets deriving fron 1 ml culture samples using the GeneElute™ total RNA Purification Kit (SIGMA), recovering it in 50 µl of Elution Solution.
|
| Label |
biotin
|
| Label protocol |
The RNAs were processed following the Affymetrix Protocol Target Labeling for Prokaryotic GeneChip® Antisense Arrays (Affymetrix Procaryotic gene Expression Manual). The protocol consists in cDNA synthesis by reverse transcription (starting with 10 µg RNA), followed by cDNA fragmentation with DNase I and labeling with Terminal Deoxynucleotidyl Transferase.
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| |
|
| Hybridization protocol |
The labeled cDNAs were then hybridised for 16h at 50°C on individual GeneChips
|
| Scan protocol |
GeneChips were washed and stained with streptavidin-conjugated phycoerythrin by using the Fluidic Station FS450 (Affymetrix) following the ProkGE-WS2v3_450 Protocol. Fluorescent images of the microarrays were acquired using a GeneChip Scanner 3000 (Affymetrix).
|
| Description |
Gene expression data from Saccharopolyspora erythraea rif6 after 72h of growth
|
| Data processing |
Raw data quality was assessed considering MAS5.0 control parameters after a global scaling at a target intensity of 100. Quality and control parameters as well as boxplot of raw intensities indicated the overall high quality of the data set and the absence of any outlying sample. Probe level data was converted to expression values using both Robust Multi-array Average procedure and MAS5.0 algorithms. In the former case, PM values have been background adjusted, normalized using invariant set normalization, and log transformed. In the latter case, intensity levels have been normalized using the Global Scaling option to target value (i.e., TGT=100).
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| |
|
| Submission date |
Jul 07, 2008 |
| Last update date |
Apr 12, 2009 |
| Contact name |
Giorgio Corti |
| E-mail(s) |
[email protected]
|
| Organization name |
National Research Council
|
| Department |
Institute of Biomedical Technologies
|
| Street address |
via Fratelli Cervi 93
|
| City |
Segrate - Milan |
| ZIP/Postal code |
20090 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6051 |
| Series (1) |
| GSE12017 |
Phenotype and expression profile of S. erythraea rifampicin-resistant (rif) mutants affected in erythromycin production |
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