|
| Status |
Public on Feb 23, 2007 |
| Title |
Hyphal Replicate #1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ACE2 knockout strain (MK106)
|
| Organism |
Candida albicans |
| Characteristics |
ACE2 knockout strain (MK106)
Hyphally induced in YPD+10% FCS @ 37C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ambion Yeast Ribopure kit
|
| Label |
Cy5
|
| Label protocol |
Twentyfive micrograms of total RNA was labeled in a total volume of 40 microlitres including 5 ng LuxA control RNA (Eurogentec), 1x first-strand buffer (Invitrogen), 0.2 pmol C. albicans-specific primer (Eurogentec), 1.5 mM (each) dATP, dTTP, and dGTP, 25 uM dCTP, 10 mM dithiothreitol, 1 ul RNasin, and 37.5 uM Cy5-dCTP (Amersham). The mixture was incubated at 65C for 5 min and then at 42C for 5 min. One microliter of RNasin (Promega) and 1 ul Superscript II reverse transcriptase (Invitrogen) were added and incubated at 42C for 1 h. Following the addition of another 1 ul of Superscript II, the incubation was continued for 1 h at 42C. The reaction was stopped by the addition of 5 mM EDTA, pH 8.0, and 0.5 M NaOH and incubation at 65C for 20 min and was neutralized by adding 0.5M acetic acid. The labeled cDNAs were purified using a QIAquick PCR purification kit (QIAGEN) and concentrated by drying under vacuum to a volume of approximately 2 ul.
|
| |
|
| Channel 2 |
| Source name |
Wildtype strain (SC5314)
|
| Organism |
Candida albicans |
| Characteristics |
Wildtype strain (SC5314)
Hyphally induced in YPD+10% FCS @ 37C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ambion Yeast Ribopure kit
|
| Label |
Cy3
|
| Label protocol |
As for Channel 1, but using 37.5 uM Cy3-dCTP (Amersham)
|
| |
|
| |
| Hybridization protocol |
Before hybridization, one of the labeled cDNA samples was resuspended in 45 ul of hybridization buffer (1x Dig Ease buffer [Roche] containing 0.5 mg ml-1 baker's tRNA and 0.5 mg ml-1 salmon sperm DNA) and then added to the second labeled cDNA. The mixture was heat denatured at 95C for 2 min, quickly cooled on ice, applied to the DNA microarray, and covered with a 24-mm by 60-mm coverslip. Slides were placed in a hybridization chamber (Corning, Palo Alto, CA) with 9 ul of 3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) in each of the wells and incubated at 37C for 16 h by immersion in a water bath. Following hybridization, the slides were immersed in 1x SSC at 50C until the coverslip fell off, washed twice for 15 min each in 1x SSC-0.1% sodium dodecyl sulfate (SDS) at 50C and once in 0.1x SSC-0.1% SDS for 20 min at 50C, and rinsed three times in 1x SSC at 50C and, finally, once in 0.2x SSC at room temperature. The slides were dried by centrifugation at 900 rpm for 5 min.
|
| Scan protocol |
The microarrays were scanned using an Axon 4000B scanner at a 10 micrometer resolution
|
| Description |
Transcriptional comparison of WT and ACE2 knockout strains grown in hyphal inducing conditions
|
| Data processing |
Low-quality spots were automatically flagged by Genepix Pro 5.0. Lowess normalization was carried out in GeneSpring.
|
| |
|
| Submission date |
Jan 15, 2007 |
| Last update date |
Feb 22, 2007 |
| 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 |
GPL3727 |
| Series (2) |
| GSE7100 |
WT vs. ACE2 knockout (grown in hyphal form) |
| GSE7105 |
Transcriptional comparison of C. albicans WT and ACE2 knockout strains |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Log2 (normalised Cy5/Cy3 ratio) |
| CH1_MEDIAN_RAW |
The median intensity value calculated from total amount of pixels of the spot in channel 1 |
| CH1_BKD_MEDIAN_RAW |
The median intensity value calculated from total amount of local background pixels of the spot in channel 1 |
| CH2_MEDIAN_RAW |
The median intensity value calculated from total amount of pixels of the spot in channel 2 |
| CH2_BKD_MEDIAN_RAW |
The median intensity value calculated from total amount of local background pixels of the spot in channel 2 |
| CH1_INTENSITY_MINUS_BKD |
Intensity minus background for channel 1 |
| CH2_INTENSITY_MINUS_BKD |
Intensity minus background for channel 2 |
| FLAGS |
Gives information about the data depending on the value of the flag. Flag 0 means unflagged. Flag -50 means spot not found when GenePix align blocks fails to align a spot indicator |
| RATIO_NORM |
Ratio of normalised intensities. The ratio is calculated by dividing channel 1 by channel 2 (Cy5/Cy3). |
