|
| Status |
Public on Nov 15, 2010 |
| Title |
Candida_albicans_UPC2_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from Candida albicans UPC2 delete TW14920, grown in YPD medium at 30 degree celsius at 1% Oxygen.
|
| Organism |
Candida albicans |
| Characteristics |
age: 3 h
growth condition: 30 degree Celsius and 1% Oxygen.
medium: YPD medium preconditioned overnight at 1% Oxygen
strain: TW14920
genotype: UPC2 deletion
|
| Growth protocol |
Overnight cultures of C. albicans TW14920 (at 21% oxygen) were diluted to an A600 of 0.2 in 50 ml of YPD medium pre-conditioned with 1% oxygen, and culture was incubated at 200 rpm at 30 degrees and 1% oxygen for 3.5 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion)
|
| 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, 2 μl Candida albicans specific RT primers mix (0.1 pmol μl (Eurogentec)) 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 Candida albicans DAY286 cells, grown in YPD medium at 30 degree celsius at 1% Oxygen.
|
| Organism |
Candida albicans |
| Characteristics |
age: 3.5 h
growth condition: 30 degree Celsius and 1% oxygen
medium: YPD medium preconditioned overnight at 1% Oxygen.
strain: DAY286
genotype: wild type
|
| Growth protocol |
Overnight cultures of C. albicans DAY286 (at 21% oxygen) were diluted to an A600 of 0.2 in 50 ml of YPD medium pre-conditioned with 1% oxygen, and culture was incubated at 200 rpm at 30 degrees and 1% oxygen for 3.5 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a RiboPure Yeast kit (Ambion)
|
| 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, 2 μl Candida albicans specific RT primers mix (0.1 pmol μl (Eurogentec)) 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 manufacture'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.
|
| Data processing |
LOWESS normalized, no background correction using the LIMMA package from Bioconductor.
|
| |
|
| Submission date |
Sep 10, 2010 |
| Last update date |
Nov 15, 2010 |
| Contact name |
Alessandro Guida |
| E-mail(s) |
alessandro.guida@ucd.ie
|
| Phone |
+353 871337884
|
| Fax |
+353 871337884
|
| Organization name |
University College Dublin
|
| Lab |
Butler group
|
| Street address |
belfield
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL10903 |
| Series (2) |
| GSE24075 |
Transcriptional profile of Candida albicans DAY286 versus UPC2 delete, both at 1% oxygen (hypoxia). |
| GSE24076 |
Hypoxia, ketoconazole, and the UPC2 gene in Candida albicans |
|
