|
| Status |
Public on Dec 07, 2012 |
| Title |
delta xprG 0 h starvation x delta xprG 12 h starvation, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
delta xprG after 0h starvation
|
| Organism |
Aspergillus nidulans |
| Characteristics |
strain/background: MH3018
genotype: delta xprG
treatment: glucose 1% during 24h followed by 0h starvation
|
| Growth protocol |
A. nidulans strains (WT, delta xprG, delta atmA) were grown on minimal medium for 24 h (0 h starvation reference), then exposed to 12 and 24 h starvation. The experiment was carried out at 37°C and 180 rpm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the RNeasy® Plant Mini Kit (Qiagen) following manufacturer's instructions, followed by DNase treatment and purification using the RNeasy® Mini Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Prior to labelling, cDNA was synthesized from 5 µg of RNA using Agilent's cDNA Master Mix. cRNA amplification and labeling were performed by adding to the samples the Agilent™ Transcription Master Mix plus Cyanine-3 (for non-starved samples) or Cyanine-5 (for starved samples) for 2 hours at 40 oC.
|
| |
|
| Channel 2 |
| Source name |
delta xprG after 12h of starvation
|
| Organism |
Aspergillus nidulans |
| Characteristics |
strain/background: MH3018
genotype: delta xprG
treatment: glucose 1% during 24h followed by 12h of starvation
|
| Growth protocol |
A. nidulans strains (WT, delta xprG, delta atmA) were grown on minimal medium for 24 h (0 h starvation reference), then exposed to 12 and 24 h starvation. The experiment was carried out at 37°C and 180 rpm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the RNeasy® Plant Mini Kit (Qiagen) following manufacturer's instructions, followed by DNase treatment and purification using the RNeasy® Mini Kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Prior to labelling, cDNA was synthesized from 5 µg of RNA using Agilent's cDNA Master Mix. cRNA amplification and labeling were performed by adding to the samples the Agilent™ Transcription Master Mix plus Cyanine-3 (for non-starved samples) or Cyanine-5 (for starved samples) for 2 hours at 40 oC.
|
| |
|
| |
| Hybridization protocol |
For the hybridization, 825 ng of each labeled cRNA was mixed with Agilent™ Fragmentation Mix and incubated at 60 °C for exactly 30 minutes to fragment RNA. The fragmentation was interrupted by adding 55 µL of 2X GE Hybridization Buffer HI-RPM. Finally, 100 µL of sample was placed down onto the microarray slide, which was mounted into the Agilent™ Microarray Hybridization Chamber Kit. The hybridization was carried out in an oven (Agilent G2545A Hybridization Oven) set to 65 °C for 17 hours. After, microarray slides were washed according to Agilent's instructions.
|
| Scan protocol |
Scanned using a GenePix® 4000B microarray scanner (Molecular Devices, USA).
|
| Description |
Biological replicate 2 of 3.
|
| Data processing |
Agilent Feature Extraction Software (v 9.5.3.1) was used for background subtraction and LOWESS normalization. After, data were processed using the TIGR platform. Data were visualized using TMeV from TIGR, and the same software has been used for statistical analysis (t test).
|
| |
|
| Submission date |
Dec 04, 2012 |
| Last update date |
Dec 07, 2012 |
| Contact name |
Nadia Graciele Krohn |
| E-mail(s) |
nadiakrohn@yahoo.com.br
|
| Organization name |
University of São Paulo
|
| Department |
Pharmacy
|
| Street address |
Av do Cafe
|
| City |
Ribeirão Preto |
| State/province |
São Paulo |
| ZIP/Postal code |
14040-903 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL15870 |
| Series (1) |
| GSE42732 |
Aspergillus nidulans: Control (0 h starvation) vs. induction of starvation (12 h and 24 h) for WT, delta xprG and delta atmA |
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