|
| Status |
Public on Mar 10, 2011 |
| Title |
Effect of Conjugated linoleic acid at 37℃ rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
90 min in Spider Media at 37℃ + 100 uM CLA
|
| Organism |
Candida albicans |
| Characteristics |
treatment: 37℃ + 100 uM CLA
|
| Treatment protocol |
C. albicans cells grown overnight in YPD at 30°C (OD600 of ~12-14) were washed in sterile distilled water and diluted to 5 X 106 cells ml-1 (OD600 of 0.1) in Spider medium supplemented with ethanol or CLA (100 μM). Cultures were incubated with shaking at either 30°C or 37°C for 90 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from quadruplicate independent biological samples using the RNeasy mini kit (Qiagen). Briefly, frozen cells were thawed out in RNeasy buffer RLT at a ratio of 3:1 [vol/vol] buffer/pellet. Resuspended cells were divided into 1 ml aliquots in 2 ml screw cap microcentrifuge tubes containing 0.6 ml of 0.5 mm diameter acid-washed glass beads. Samples were homogenized 6 times, for 5 minutes each, in a BeadBeater set at maximum speed.
|
| Label |
Cy5
|
| Label protocol |
20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of Cy5-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
90 min in Spider Media at 30℃
|
| Organism |
Candida albicans |
| Characteristics |
treatment: 30℃
|
| Treatment protocol |
C. albicans cells grown overnight in YPD at 30°C (OD600 of ~12-14) were washed in sterile distilled water and diluted to 5 X 106 cells ml-1 (OD600 of 0.1) in Spider medium supplemented with ethanol or CLA (100 μM). Cultures were incubated with shaking at either 30°C or 37°C for 90 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from quadruplicate independent biological samples using the RNeasy mini kit (Qiagen). Briefly, frozen cells were thawed out in RNeasy buffer RLT at a ratio of 3:1 [vol/vol] buffer/pellet. Resuspended cells were divided into 1 ml aliquots in 2 ml screw cap microcentrifuge tubes containing 0.6 ml of 0.5 mm diameter acid-washed glass beads. Samples were homogenized 6 times, for 5 minutes each, in a BeadBeater set at maximum speed.
|
| Label |
Cy3
|
| Label protocol |
20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of Cy5-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
The microarray slides were pre-hybridized for 2 hours at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. Hybridization was done with an Advalytix SlideBooster. The slides were washed twice with SSC-0.2% SDS, once with 0.1X SSC-0.2% SDS and 3 times with 0.1X SSC. A final wash with isopropanol was performed and slides were air-dried.
|
| Scan protocol |
Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 10-µm resolution.
|
| Data processing |
Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).
|
| |
|
| Submission date |
Dec 02, 2010 |
| Last update date |
Mar 10, 2011 |
| Contact name |
Andre Nantel |
| E-mail(s) |
andre.nantel@nrc-cnrc.gc.ca
|
| Phone |
514-496-6370
|
| Fax |
514-496-9127
|
| Organization name |
National Research Council of Canada
|
| Department |
Biotechnology Research Institute
|
| Street address |
6100 Royalmount
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H4P 2R2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL9818 |
| Series (1) |
| GSE25822 |
Conjugated linoleic acid inhibits hyphal growth in Candida albicans by modulating Ras1 cellular levels and down-regulating TEC1 expression |
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