|
| Status |
Public on Jun 18, 2013 |
| Title |
Apm_5minNi_rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Acidiphilium sp. PM_untreated
|
| Organism |
Acidiphilium sp. PM |
| Characteristics |
treatment: Not exposed to Ni
|
| Treatment protocol |
In early exponential phase, 10mM Ni was added to twelve 100-ml cultures. Six of them were harvested after 5 min, and the remaining six after 30min. Six other 100-ml cultures were left untreated and harvested after 30min. After centrifugation, cells were snap-frozen and stored at -80ºC until RNA extraction.
|
| Growth protocol |
Acidiphilium sp. PM was grown aerobically at 30ºC in media described elsewhere (San Martin-Uriz et al, 2013. Submitted).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol Plus RNA purification system (Ambion), which includes an on-column DNase treatment. After elution, RNA was treated again with TURBO DNase (Ambion).
|
| Label |
Cy3
|
| Label protocol |
20 µg of total RNA were retrotranscribed using random hexamers, a mixture of aminoallyl-dUTP and dNTPs and Superscript® III RNAse H- (Invitrogen). RNA was then degraded at 70ºC in the presence of 100mM NaOH/10mM EDTA. Purified aminoallyl-cDNAs were then incubated with DMSO-dissolved Cyanine-based dyes for labelling. Labelled cDNAs were purified with PCR purification kit (Qiagen) prior to hybridization.
|
| |
|
| Channel 2 |
| Source name |
Acidiphilium sp. PM_10mM Ni_5min
|
| Organism |
Acidiphilium sp. PM |
| Characteristics |
treatment: Exposed to 10mM Ni for 5 min
|
| Treatment protocol |
In early exponential phase, 10mM Ni was added to twelve 100-ml cultures. Six of them were harvested after 5 min, and the remaining six after 30min. Six other 100-ml cultures were left untreated and harvested after 30min. After centrifugation, cells were snap-frozen and stored at -80ºC until RNA extraction.
|
| Growth protocol |
Acidiphilium sp. PM was grown aerobically at 30ºC in media described elsewhere (San Martin-Uriz et al, 2013. Submitted).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol Plus RNA purification system (Ambion), which includes an on-column DNase treatment. After elution, RNA was treated again with TURBO DNase (Ambion).
|
| Label |
Cy5
|
| Label protocol |
20 µg of total RNA were retrotranscribed using random hexamers, a mixture of aminoallyl-dUTP and dNTPs and Superscript® III RNAse H- (Invitrogen). RNA was then degraded at 70ºC in the presence of 100mM NaOH/10mM EDTA. Purified aminoallyl-cDNAs were then incubated with DMSO-dissolved Cyanine-based dyes for labelling. Labelled cDNAs were purified with PCR purification kit (Qiagen) prior to hybridization.
|
| |
|
| |
| Hybridization protocol |
Spots in the microarray were hydrated and blocked at 42ºC prior to hybridization in a mixture containing 1x HybIt Hybridization Solution, 75ng/µl Herring sperm ssDNA and Cy3-/Cy5-labelled cDNAs. Hybridized arrays were incubated overnight at 65ºC .
|
| Scan protocol |
Hybridized microarrays were scanned and analyzed for Cy3 (532 nm) and Cy5 (635 nm) dyes with a GenePix 4100A scanner (Axon Instruments, Foster City, CA) setting a saturation tolerance of 0.005%.
|
| Data processing |
Local feature background median was substracted from raw data of average fluorescence intensity values and exported to AlmaZen (BioAlma, Tres Cantos, Spain). Normalization was performed applying LOWESS algorithm individually to each of the blocks in the microarray (LOWESS per pin). Comparative analysis consisted of a paired t-test (p<0.05). The criteria to include spots in the analysis were: (i) average signal intensity >2000 FU and (ii) 0.50 > fold change >2 for the experiments comparing 0 vs 5 min exposures to 10mM Ni or 0,29 > fold change > 3 for the experiments comparing 0 vs 30 min exposures to 10mM Ni.
|
| |
|
| Submission date |
Jun 17, 2013 |
| Last update date |
Jun 18, 2013 |
| Contact name |
Patxi San Martin-Uriz |
| E-mail(s) |
sanmartinup@cab.inta-csic.es
|
| Organization name |
Instituto Nacional de Tecnica Aeroespacial
|
| Department |
Centro de Astrobiologia
|
| Street address |
Ctra. de Ajalvir, km. 4
|
| City |
Torrejon de Ardoz |
| State/province |
Madrid |
| ZIP/Postal code |
28850 |
| Country |
Spain |
| |
|
| Platform ID |
GPL17306 |
| Series (1) |
| GSE48042 |
Transcriptome analysis of the early response to nickel (Ni) in Acidiphilium sp. PM |
|
