|
| Status |
Public on Nov 30, 2011 |
| Title |
Candida_parapsilosis_normoxia_vs_hypoxia_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from C. parapsilosis CLIB214 grown in YPD medium at 30 degree celsius and 21% oxygen (normoxia)
|
| Organism |
Candida parapsilosis |
| Characteristics |
age: 3.5 h
growth: 30 degree Celsius and 21% Oxygen.
medium: YPD medium
strain: CLIB214
|
| Growth protocol |
Overnight cultures in YPD at 30 degree Celsius were diluted to an A600 of 0.2 in 250 ml YPD. Cultures were incubated at 200 RPM at 30 C for about 3.5 hours in YPD until reaching an A600 of 0.6. Cultures were then split into two groups, spun down and resuspended in 100 ml YPD media. One culture was incubated at 30 C, 200 RPM, 21 % O2, and the other one at 30 degree Celsius, 200 RPM, 1 % O2. Media for hypoxic growth had been preincubated in the hypoxic chamber overnight. Cells were grown for an additional 2 hours. Cells were pelleted and frozen in RNA Later at -80 degree Celsius.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using a RiboPure Yeast kit (Ambion). RNA concentration was checked using Nanodrop. RNA quality was checked using Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
RNA was concentrated and readjusted to a concentration of 4 μg/μl with RNase free water. A mixture of 24 μg RNA, 100 pmol of oligo dT primers (Invitrogen) and RNase free water to a volume of 18.5 μl was incubated at 70 degrees Celsius for 10 min. Tubes were then placed on ice briefly to stop the reaction and centrifuged briefly in order to collect the contents of the tube. The cDNA synthesis step was carried out in dim light conditions because of the photosensitive properties of the Cyanine-3 (Cy3) dye. A mixture of 8 μll 5X First Strand buffer, 4 μll 0.1 M Dithiothreitol (DTT), 3 μl 20 mM dNTP mix - dCTP (consisting of 6.67 mM each dATP, dGTP, and dTTP), 1 μl 2 mM dCTP and 2 μl (400 U) of Superscript II Reverse Transcriptase was added to the tubes. 2 μl of 1 mM Cy3 (Cy3-dCTP from Amersham Biosciences, 25 nmol) or Cy5 (Cy5-dCTP from Amersham Biosciences, 25 nmol) was added to the relevant tubes. Tubes were lightly mixed and centrifuged briefly in order to collect the contents of the tube. After 2 hours incubation at 42 degrees Celsius, the tubes were given a quick spin and another 2 μl (400 U) Superscript II Reverse Transcriptase was added. The reaction was incubated for an additional 1 hour at 42 degrees Celsius. Again, tubes were then placed on ice briefly to stop the reaction and centrifuged briefly in order to collect the contents of the tube. Any remaining RNA was degraded by the addition of 1 μl NRase A (0.05 mg ml-1) and 1 μl NRase H (0.05 units ml-1) and incubated at 37 degrees Celsius for 30 min.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from C. parapsilosis CLIB214 grown in YPD medium at 30 degree celsius and 1% oxygen (hypoxia)
|
| Organism |
Candida parapsilosis |
| Characteristics |
age: 3.5 h
growth: 30 degree Celsius and 1% Oxygen.
medium: YPD medium preconditioned overnight at 1% Oxygen.
strain: CLIB214
|
| Growth protocol |
Overnight cultures in YPD at 30 degree Celsius were diluted to an A600 of 0.2 in 250 ml YPD. Cultures were incubated at 200 RPM at 30 C for about 3.5 hours in YPD until reaching an A600 of 0.6. Cultures were then split into two groups, spun down and resuspended in 100 ml YPD media. One culture was incubated at 30 C, 200 RPM, 21 % O2, and the other one at 30 degree Celsius, 200 RPM, 1 % O2. Media for hypoxic growth had been preincubated in the hypoxic chamber overnight. Cells were grown for an additional 2 hours. Cells were pelleted and frozen in RNA Later at -80 degree Celsius.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using a RiboPure Yeast kit (Ambion). RNA concentration was checked using Nanodrop. RNA quality was checked using Agilent Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
RNA was concentrated and readjusted to a concentration of 4 μg/μl with RNase free water. A mixture of 24 μg RNA, 100 pmol of oligo dT primers (Invitrogen) and RNase free water to a volume of 18.5 μl was incubated at 70 degrees Celsius for 10 min. Tubes were then placed on ice briefly to stop the reaction and centrifuged briefly in order to collect the contents of the tube. The cDNA synthesis step was carried out in dim light conditions because of the photosensitive properties of the Cyanine-3 (Cy3) dye. A mixture of 8 μll 5X First Strand buffer, 4 μll 0.1 M Dithiothreitol (DTT), 3 μl 20 mM dNTP mix - dCTP (consisting of 6.67 mM each dATP, dGTP, and dTTP), 1 μl 2 mM dCTP and 2 μl (400 U) of Superscript II Reverse Transcriptase was added to the tubes. 2 μl of 1 mM Cy3 (Cy3-dCTP from Amersham Biosciences, 25 nmol) or Cy5 (Cy5-dCTP from Amersham Biosciences, 25 nmol) was added to the relevant tubes. Tubes were lightly mixed and centrifuged briefly in order to collect the contents of the tube. After 2 hours incubation at 42 degrees Celsius, the tubes were given a quick spin and another 2 μl (400 U) Superscript II Reverse Transcriptase was added. The reaction was incubated for an additional 1 hour at 42 degrees Celsius. Again, tubes were then placed on ice briefly to stop the reaction and centrifuged briefly in order to collect the contents of the tube. Any remaining RNA was degraded by the addition of 1 μl NRase A (0.05 mg ml-1) and 1 μl NRase H (0.05 units ml-1) and incubated at 37 degrees Celsius for 30 min.
|
| |
|
| |
| Hybridization protocol |
Purified cDNA was denatured at 95 degree Celsius 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 instruction.
|
| 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.
|
| Data processing |
LOWESS normalized, no background correction using the LIMMA package from Bioconductor.
|
| |
|
| Submission date |
Oct 10, 2011 |
| Last update date |
Nov 30, 2011 |
| 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 |
GPL13192 |
| Series (2) |
| GSE32712 |
Transcriptional profile of Candida parapsilosis CLIB214 21% Oxygen (normoxia) versus at 1% oxygen (hypoxia). |
| GSE32716 |
Transcriptional landscape of the pathogenic yeast Candida parapsilosis |
|
