|
| Status |
Public on Dec 11, 2010 |
| Title |
18 hours antibody C7 exposure 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
candida not exposed
|
| Organism |
Candida albicans |
| Characteristics |
strain: SC5314
growth conditions: maintained on solid YPD medium and incubated at 30ºC
|
| Treatment protocol |
Monoclonal antibody C7 was added at 12’5 ug/ml final concentration in order to inhibit fungal growth and avoiding fungicidal effect
|
| Growth protocol |
A saturated culture of C. albicans SC5314 grown overnight in modified Lee’s medium was diluted to an optical density at 600 nm of approximately 0.1 and split in two aliquots. Monoclonal antibody C7 was added to one of the cultures at 12.5 µg/ml final concentration whereas the second one remained untreated. Both cultures were incubated for 18 h at 37ºC before harvesting cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by centrifugation, transferred to a pre-cooled teflon vessel and quickly chilled in liquid nitrogen. Cells were disrupted by shaking with a tungsten carbide bead at 2000 rpm for 2 minutes in a micro-dismembrator and RNA was extracted with Trizol Reagent.
|
| Label |
Cy5
|
| Label protocol |
Fifteen micrograms of total RNA was labeled in a total volume of 40 µl including 1x first-strand buffer (Invitrogen), 0.2 pmol C. albicans-specific primer mix (Eurogentec), 1.5 mM (each) dATP, dTTP, and dGTP, 25 µM dCTP, 10 mM dithiothreitol, 1 µl RNasin, and 37.5 µM Cy5-dCTP or Cy3-dCTP (Amersham). The mixture was incubated at 65°C for 5 min and then at 42°C for 5 min. One microliter of RNasin (Promega) and 1 µl Superscript II reverse transcriptase (Invitrogen) were added and incubated at 42°C for 1.5 h. Following the addition of another 1 µl of Superscript II, the incubation was continued for 1.5 h at 42°C. The reaction was stopped by the addition of 5 mM EDTA, pH 8.0, and 0.5 M NaOH and incubation at 65°C for 20 min and was neutralized by adding 0.5 M acetic acid. The labeled cDNAs were purified using a QIAquick PCR purification kit (QIAGEN) and concentrated by filtration through microcon columns (Millipore) to a volume of 10 µl. The amount of cDNA as well as the incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified on a spectrophotometer using Ultra-micro cuvettes.
|
| |
|
| Channel 2 |
| Source name |
candida exposed to antibody C7
|
| Organism |
Candida albicans |
| Characteristics |
strain: SC5314
growth conditions: maintained on solid YPD medium and incubated at 30ºC
|
| Treatment protocol |
Monoclonal antibody C7 was added at 12’5 ug/ml final concentration in order to inhibit fungal growth and avoiding fungicidal effect
|
| Growth protocol |
A saturated culture of C. albicans SC5314 grown overnight in modified Lee’s medium was diluted to an optical density at 600 nm of approximately 0.1 and split in two aliquots. Monoclonal antibody C7 was added to one of the cultures at 12.5 µg/ml final concentration whereas the second one remained untreated. Both cultures were incubated for 18 h at 37ºC before harvesting cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by centrifugation, transferred to a pre-cooled teflon vessel and quickly chilled in liquid nitrogen. Cells were disrupted by shaking with a tungsten carbide bead at 2000 rpm for 2 minutes in a micro-dismembrator and RNA was extracted with Trizol Reagent.
|
| Label |
Cy3
|
| Label protocol |
Fifteen micrograms of total RNA was labeled in a total volume of 40 µl including 1x first-strand buffer (Invitrogen), 0.2 pmol C. albicans-specific primer mix (Eurogentec), 1.5 mM (each) dATP, dTTP, and dGTP, 25 µM dCTP, 10 mM dithiothreitol, 1 µl RNasin, and 37.5 µM Cy5-dCTP or Cy3-dCTP (Amersham). The mixture was incubated at 65°C for 5 min and then at 42°C for 5 min. One microliter of RNasin (Promega) and 1 µl Superscript II reverse transcriptase (Invitrogen) were added and incubated at 42°C for 1.5 h. Following the addition of another 1 µl of Superscript II, the incubation was continued for 1.5 h at 42°C. The reaction was stopped by the addition of 5 mM EDTA, pH 8.0, and 0.5 M NaOH and incubation at 65°C for 20 min and was neutralized by adding 0.5 M acetic acid. The labeled cDNAs were purified using a QIAquick PCR purification kit (QIAGEN) and concentrated by filtration through microcon columns (Millipore) to a volume of 10 µl. The amount of cDNA as well as the incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified on a spectrophotometer using Ultra-micro cuvettes.
|
| |
|
| |
| Hybridization protocol |
A mixture containing 5µl each Cy3 and Cy5-labeled cDNA samples plus 5µl herring sperm DNA (10 mg/ml) was heat denatured at 95°C for 2 min and quickly cooled on ice. Forty microliters of hybridization buffer were added to the DNA probe, and the solution was placed onto DNA microarrays slides, which were covered with a rimmed cover glass (Erie Scientific, Portsmouth, NH). Slides were placed in a hybridization chamber (Corning, Palo Alto, CA) and hybridized for 24 h at 42°C by immersion in a water bath. Following hybridization, the slides were immersed in 2x SSC, 1% SDS for 15 min. and washed twice for 5 min each in 1x SSC at room temperature. The slides were dried by centrifugation at 1100 rpm for 5 min. Four dye-swap comparative analysis were performed.
|
| Scan protocol |
The microarrays were scanned using a GenePix4000B microarray scanner (Axon Instruments) at a 10-µm resolution. GenePix Pro 4.0 analysis software (Axon Instruments) was used to locate spots in the microarray with the appropriate grid and to obtain the two Cy3/Cy5 image TIFF files. All images were further processed using GenePix 4.0 software according to manufacturer instructions. Low-quality spots were automatically flagged by Genepix Pro 4.0.
|
| Description |
Biological replicate 2 of 4
|
| Data processing |
Data were evaluated with the GeneSpring version 5.0 software (Silicon Genetics, Redwood City, CA) and normalized in a per-chip (to the 50 percentile) and per-gene (to the median) way. Median F635, F532, B635, and B532 of each spot were loaded into GeneSpring for calculation of signal and background intensities. Signals were log transformed and per-chip normalized by an intensity-dependent method (Lowess) applied to the print-tip region.
|
| |
|
| Submission date |
Dec 09, 2010 |
| Last update date |
Dec 11, 2010 |
| Contact name |
Iñigo Fernandez de Larrinoa |
| E-mail(s) |
if.larrinoa@ehu.es
|
| Phone |
+34 943 218 212
|
| Fax |
+34 943 015 270
|
| Organization name |
Universidad del País Vasco (UPV/EHU)
|
| Department |
Química Aplicada
|
| Lab |
Bioquímica
|
| Street address |
Pº Manuel de Lardizabal #3
|
| City |
San Sebastian |
| State/province |
Guipuzcoa |
| ZIP/Postal code |
E-20.018 |
| Country |
Spain |
| |
|
| Platform ID |
GPL3727 |
| Series (1) |
| GSE25969 |
Eighteen hours monoclonal antibody C7 exposure |
|
