|
| Status |
Public on Jan 15, 2009 |
| Title |
Candida_parapsilosis_Normoxia_Vs_Hypoxia_rep4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Candida parapsilosis cells grown in hypoxic conditions (1% oxygen) in SD medium
|
| Organism |
Candida parapsilosis |
| Characteristics |
Age: 3 h
Growth condition: hypoxic
Medium: SD
Strain: CLIB214
|
| Growth protocol |
Overnight cultures of C. parapsilosis CLIB214 were diluted to a A600 of 0.2 in 300 ml of SD at 37°C in normoxic (atmosphere oxygen) conditions for 3 hours. Two 100 ml aliquots were removed, the cells were collected by centrifugation, and re-suspended in media pre-conditioned at the relevant concentration of oxygen. One flask was incubated in normoxic conditions (21% oxygen) in a standard orbital shaker, and the other was incubated in hypoxic conditions (1% 02, 99% N2) in a dedicated chamber (In vivo 400 workstation). Both cultures were incubated at 37°C at 200 rpm. Ch1 represents the hypoxic sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion).
|
| Label |
Cy5
|
| Label protocol |
Twenty-four micrograms of total RNA was used for cDNA labeling. Briefly RNA samples were incubated with 100 pmol of anchor oligonucleotide deoxyriboslthymine (Invitrogen) at 70°C for 10 min, after which following reaction reagents were added including 1 X First strand buffer, 6.67 mM dATP, dTTP, and dGTP; 2mM dCTP; 100 mM dithiothreitol; 2 µl Superscript II reverse transcriptase (Invitrogen). 37.5 µM Cy5-dCTP were then added into the mixture in dark. The mixture were left at 42°C for two hours followed by adding additional 2 µl Superscript II reverse transcriptase, and reaction was continued for one more hour. 50 µg ml-1 RNase A and 0.05 unit µl RNase H were added and the reaction was incubated for 30 min at 37°C.
|
| |
|
| Channel 2 |
| Source name |
Candida parapsilosis cells grown in normoxic conditions (21% oxygen) in SD medium
|
| Organism |
Candida parapsilosis |
| Characteristics |
Age: 3 h
Growth condition: normoxic
Medium: SD
Strain: CLIB214
|
| Growth protocol |
Overnight cultures of C. parapsilosis CLIB214 were diluted to a A600 of 0.2 in 300 ml of SD at 37°C in normoxic (atmosphere oxygen) conditions for 3 hours. Two 100 ml aliquots were removed, the cells were collected by centrifugation, and re-suspended in media pre-conditioned at the relevant concentration of oxygen. One flask was incubated in normoxic conditions (21% oxygen) in a standard orbital shaker, and the other was incubated in hypoxic condtions (1% 02, 99% N2) in a dedicated chamber (In vivo 400 workstation). Both cultures were incubated at 37°C at 200 rpm. Ch2 represents the normoxic sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion).
|
| Label |
Cy3
|
| Label protocol |
Twenty-four micrograms of total RNA was used for cDNA labeling. Briefly RNA samples were incubated with 100 pmol of anchor oligonucleotide deoxyriboslthymine (Invitrogen) at 70°C for 10 min, after which following reaction reagents were added including 1 X First strand buffer, 6.67 mM dATP, dTTP, and dGTP; 2mM dCTP; 100 mM dithiothreitol; 2 µl Superscript II reverse transcriptase (Invitrogen). 37.5 µM Cy3-dCTP were then added into the mixture in dark. The mixture were left at 42°C for two hours followed by adding additional 2 µl Superscript II reverse transcriptase, and reaction was continued for one more hour. 50 µg ml-1 RNase A and 0.05 unit µl RNase H were added and the reaction was incubated for 30 min at 37°C.
|
| |
|
| |
| Hybridization protocol |
Purified cDNA was denatured at 95 °C for 2 min, and then chilled on ice for 10 sec. Two cDNA samples were mixed. The hybridization was carried out using Agilent Gene Expression Hybridization Kit (Agilent Technologies) according to the manufacturer’s manual.
|
| 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 4 of 6.
|
| 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) |
geraldine.butler@ucd.ie
|
| 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 |
GPL7693 |
| Series (2) |
| GSE13722 |
Transcriptional response of Candida parapsilosis in low oxygen (hypoxic) conditions in SD media |
| GSE13832 |
Hypoxia and biofilms in Candida parapsilosis |
|
