|
| Status |
Public on Jan 15, 2009 |
| Title |
Candida_parapsilosis_Biofilm_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Planktonic Candida parapsilosis cells grown in SD medium
|
| Organism |
Candida parapsilosis |
| Characteristics |
Planktonic cells
Medium: SD
Strain: CLIB214
|
| Growth protocol |
Flasks containing 50 ml of the same medium used for the biofilm were inoculated at A600 of 0.2 from an overnight culture. Cells were grown at 37°C with orbital shaking to an A600 of 1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using an RNeasy kit (Qiagen). The quality and concentration of the isolated RNA was analyzed using an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Reverse transcription, cDNA labeling and probe purification were carried out using 1 an Atlas Powerscript Fluoresecent labeling kit (Takara) according to the manufacturer’s instructions, starting with 5 µg of total RNA.
|
| |
|
| Channel 2 |
| Source name |
Candida parapsilosis biofilm cells grown in SD medium
|
| Organism |
Candida parapsilosis |
| Characteristics |
Biofilm cells
Medium: SD
Strain: CLIB214
|
| Growth protocol |
Biofilm growth in continuous flow was carried out in a microfermentor system as described in Garcia-Sanchez et al. {, 2004}. Briefly, this consists of a glass vessel with a 40 ml incubation chamber with tubes allowing entry and evacuation and continuous medium flow. Air under pressure is supplied to allow medium to flow through the chamber. The continuous flow allows cells to grow as a biofilm on a slide positioned in the chamber and minimizes planktonic growth. Thermanox slides (Nunc) were first immersed in an inoculum of the appropriate culture at A600 of 1 for 1 h at 37°C to allow adherence, and were then transferred to the microfermentor system. Experiments were performed at 37°C and biofilm was sampled after 50h growth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using an RNeasy kit (Qiagen). The quality and concentration of the isolated RNA was analyzed using an Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Reverse transcription, cDNA labeling and probe purification were carried out using 1 an Atlas Powerscript Fluoresecent labeling kit (Takara) according to the manufacturer’s instructions, starting with 5 µg of total RNA.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were prehybridized with in 5X SSC, 0.1% SDS, 10 mg ml-1 BSA for 1 h at 42ºC, washed 3 times in water, 1 time in isopropanol and dried. Incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified by measuring the absorbance of each sample at 260 nm, 550 nm or 650 nm. Samples were used if incorporation was greater than 15 pmol. The labeled cDNAs were concentrated by drying under vacuum, resuspended and pooled in 45 ul of hybridization buffer (1X DigEasy (Roche) containing 0.5 mg ml-1 bakers yeast tRNA (Invitrogen) and 0.5 mg ml-1 salmon sperm DNA (Sigma)). The mixture was denatured at 95ºC for 2 min, quickly cooled on ice, applied to the DNA microarray and covered with a 24 mm x 60 mm coverslip. Slides were placed in a hybridization chamber (Corning, Palo Alto, CA) and incubated at 42ºC for approximately 16 h. The slides were washed in 1 X SSC at 42ºC until the coverslip fell off, and then washed 3 times for 10 min in 1 X SSC, 0.1% SDS at 42ºC, 3 times 5 min in 1 X SSC at 42ºC, and finally rinsed in 0.2 X SSC at room temperature. The slides were dried by centrifugation at 1000 rpm for 5 min.
|
| Scan protocol |
Scanned with an Axon 4000B scanner at 10 µm resolution. Data was acquired using GenePix 5.0 software.
The quality and concentration of the isolated RNA was analyzed using an Agilent 2100 Bioanalyzer.
|
| Description |
Biological replicate 2 of 4.
|
| Data processing |
LOWESS normalized, no background correction using the LIMMA package from Bioconductor.
|
| |
|
| Submission date |
Nov 24, 2008 |
| Last update date |
Dec 04, 2008 |
| Contact name |
Geraldine Butler |
| E-mail(s) |
[email protected]
|
| Organization name |
University Colege Dublin Conway Institute
|
| Department |
School of Biomolecular and Biomedical Science
|
| Lab |
Butler lab
|
| Street address |
Belfield
|
| City |
Dublin |
| ZIP/Postal code |
D4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL4574 |
| Series (2) |
| GSE13717 |
Transcriptional profile of Candida parapsilosis in SD media |
| GSE13832 |
Hypoxia and biofilms in Candida parapsilosis |
|
