|
| Status |
Public on Feb 23, 2007 |
| Title |
Hyphal Replicate #3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ACE2 knockout strain (MK106) hyphal
|
| 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 |
Twenty-five micrograms of total RNA was labeled in a total volume of 40 µl 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 µM dCTP, 10 mM dithiothreitol, 1 µl RNasin, and 37.5 µM Cy5-dCTP (Amersham). The mixture was incubated at 65°C for 5 min and then at 42°C for 5 min. One microliter of RNasin (Promega) and 1 µl Superscript II reverse transcriptase (Invitrogen) were added and incubated at 42°C for 1 h. Following the addition of another 1 µl of Superscript II, the incubation was continued for 1 h at 42°C. The reaction was stopped by the addition of 5 mM EDTA, pH 8.0, and 0.5 M NaOH and incubation at 65°C for 20 min and was neutralized by adding 0.5 M 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 µl.
|
| |
|
| Channel 2 |
| Source name |
Wildtype strain (SC5314) hyphal
|
| 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 µM Cy3-dCTP (Amersham)
|
| |
|
| |
| Hybridization protocol |
Before hybridization, one of the labeled cDNA samples was resuspended in 45 µl 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 95°C 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 µl of 3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) in each of the wells and incubated at 37°C for 16 h by immersion in a water bath. Following hybridization, the slides were immersed in 1x SSC at 50°C until the coverslip fell off, washed twice for 15 min each in 1x SSC-0.1% sodium dodecyl sulfate (SDS) at 50°C and once in 0.1x SSC-0.1% SDS for 20 min at 50°C, and rinsed three times in 1x SSC at 50°C 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-µm 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). |
