|
| Status |
Public on Apr 02, 2009 |
| Title |
delta crzA time 30 min vs wild type time 30 min upon 200 mM CaCl2 dye swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
delta crzA conidia grown for 16 hours plus 30 min upon 200 mM CaCl2
|
| Organism |
Aspergillus fumigatus |
| Characteristics |
genotype/variation: delta crzA
agent: CaCl2
time: 30 minutes
|
| Biomaterial provider |
total RNA
|
| Treatment protocol |
For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus delta crzA strain were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C. After this time, the cultures from the wild type and mutant strains were added by calcium chloride (200 mM) and incubated for 10 and 30 more minutes before centrifugation and RNA extraction.
|
| Growth protocol |
For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus delta crzA strain were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At each time point the micelia was harvested by centrifugation and quickly frozen in liquid nitrogen. For total RNA isolation, the micelia weas disrupted by grinding in liquid nitrogen and total RNA was extracted with Trizol reagent (Invitrogen). Ten micrograms of RNA from each treatment were then fractionated in 2.2 M formaldehyde, 1.2% agarose gel, stained with ethidium bromide, and then visualized with UV-light. The presence of intact 25S and 17S ribosomal RNA bands was used as a criterion to assess the integrity of the RNA.
|
| Label |
Cy3
|
| Label protocol |
The RNA samples were extracted with Trizol and further purified with the RNeasy kit (Qiagen, Germany) and directly labelled by incorporation of Cy3-dUTP (GE Health Care). Briefly, 30 µg of total RNA were mixed with 20 µg of Randon Primer Hexamers (GE Health Care), 100 pmol of oligo dTV (Invitrogen) and 40 U of RNAse Out (Invitrogen) in a volume of 13 µl and heated to 70°C for 5 minutes. The tubes were briefly chilled to 4°C for 5 minutes and mixed with 5 µl of 5X first-strand buffer (Invitrogen), 2 µl DTT (Invitrogen), 2 µl dNTP (5 mM dATP, 5 mM dCTP, 5 mM dGTP, 2 mM dTTP), 2 µl of Suprescript III (Invitrogen) and 1 µl of Cy3-dUTP (25 nmol). The reaction was then incubated at 25°C for 5 minutes and at 42°C for 3 hours. The RNA was degraded by adding 2.5 µl EDTA (0.5 M, pH 8) and 5 µl NaOH (1M) following an incubation at 37°C for 40 minutes. The resulting first strand cDNA was purified and concentrated using a Microcon YM-50 cartridge.
|
| |
|
| Channel 2 |
| Source name |
wild type conidia grown for 16 hours plus 30 min upon 200 mM CaCl2
|
| Organism |
Aspergillus fumigatus |
| Characteristics |
genotype/variation: wild type
agent: CaCl2
time: 30 minutes
|
| Biomaterial provider |
total RNA
|
| Treatment protocol |
For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus wild type (CEA17 DakuBKU80) strain were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C. After this time, the cultures from the wild type and mutant strains were added by calcium chloride (200 mM) and incubated for 10 and 30 more minutes before centrifugation and RNA extraction.
|
| Growth protocol |
For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus wild type (CEA17 DakuBKU80) strain were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At each time point the micelia was harvested by centrifugation and quickly frozen in liquid nitrogen. For total RNA isolation, the micelia weas disrupted by grinding in liquid nitrogen and total RNA was extracted with Trizol reagent (Invitrogen). Ten micrograms of RNA from each treatment were then fractionated in 2.2 M formaldehyde, 1.2% agarose gel, stained with ethidium bromide, and then visualized with UV-light. The presence of intact 25S and 17S ribosomal RNA bands was used as a criterion to assess the integrity of the RNA.
|
| Label |
Cy5
|
| Label protocol |
The RNA samples were extracted with Trizol and further purified with the RNeasy kit (Qiagen, Germany) and directly labelled by incorporation of Cy3-dUTP (GE Health Care). Briefly, 30 µg of total RNA were mixed with 20 µg of Randon Primer Hexamers (GE Health Care), 100 pmol of oligo dTV (Invitrogen) and 40 U of RNAse Out (Invitrogen) in a volume of 13 µl and heated to 70°C for 5 minutes. The tubes were briefly chilled to 4°C for 5 minutes and mixed with 5 µl of 5X first-strand buffer (Invitrogen), 2 µl DTT (Invitrogen), 2 µl dNTP (5 mM dATP, 5 mM dCTP, 5 mM dGTP, 2 mM dTTP), 2 µl of Suprescript III (Invitrogen) and 1 µl of Cy3-dUTP (25 nmol). The reaction was then incubated at 25°C for 5 minutes and at 42°C for 3 hours. The RNA was degraded by adding 2.5 µl EDTA (0.5 M, pH 8) and 5 µl NaOH (1M) following an incubation at 37°C for 40 minutes. The resulting first strand cDNA was purified and concentrated using a Microcon YM-50 cartridge.
|
| |
|
| |
| Hybridization protocol |
Labelled cDNAs were mixed with 8 µl Liquid block RPN3601 Batch 24 (GE Health Care), 5.5 µl SDS 2% and 19 µl SSC (20X) and the volume adjusted to 110 µl. Slides were hybridized overnight (42°C) in a Gene-Tac Hybridization Station (Genomic Solutions, Inc.) and washed in 2x SSC 0,5% SDS, 0,5x SSC and 0.05x SSC. All washing steps consisted of 1 min of flow, followed by 5 min of incubation
|
| Scan protocol |
Slides were dried by centrifugation and subjected to fluorescent detection with a GMS 418 Array Scanner (Affymetrix Inc., Santa Clara, CA).
|
| Description |
This hybridization used the wild type RNA from a 16 hours culture plus 30 minutes upon calcium chloride (200 mM) treatment as reference. The test RNA was obtained from a delta crzA mutant strain 16 hours culture treated for 30 minutes with 200 mM calcium chloride.
|
| Data processing |
TIFF images generated were analyzed using TIGR Spotfinder (http://www.tigr.org/software/) to obtain relative transcript levels. Spotfinder uses a dynamic thresholding algorithm to identify spots and calculate local background and integrated fluorescence intensity for each of the Cy3 and Cy5 channels. This software was also useful in eliminating questionable spots (those for which shape deviates significantly from that of a true circle, or those with a low signal-to-noise ratio). Spots with very low intensity in one channel (and thus aberrantly high or low expression ratios) were removed and eliminated from future analyses. Data was normalized using a local regression technique LOWESS (LOcally WEighted Scatterplot Smoothing) for hybridizations with RNA-based samples and SD regularization of the Cy5/Cy3 ratio across all sectors (blocks) of the array using a software tool MIDAS (http://www.tigr.org/softlab). Following normalization, the values for each gene's in-slide replicates were averaged (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects. Differentially expressed genes at the 95% confidence level were determined using intensity-dependent Z-scores (with Z=1.96) as implemented in MIDAS and the union of all genes identified at each time point were considered significant in this experiment. The resulting data were organized and visualized based on similar expression vectors in genes using Euclidean distance and hierarchical clustering with average linkage clustering method to view the whole data set and k-means to group the genes in clusters with TIGR MEV (multi experiment viewer), also available at http://www.tigr.org/software.
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| |
|
| Submission date |
Mar 26, 2009 |
| Last update date |
Apr 01, 2009 |
| Contact name |
Gustavo Henrique Goldman |
| E-mail(s) |
ggoldman@usp.br
|
| Phone |
55-016-36024280
|
| Fax |
55-016-36024280
|
| Organization name |
Faculty of Pharmaceutical Sciences of Ribeirao Preto
|
| Department |
Departament of Pharmaceutical Sciences
|
| Lab |
Laboratory of Molecular Biology
|
| Street address |
Avenida do Café, s/n
|
| City |
Ribeirao Preto |
| State/province |
Sao Paulo |
| ZIP/Postal code |
14040-903 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL8346 |
| Series (1) |
| GSE15432 |
Transcriptome analysis of Aspergillus fumigatus delta crzA mutant upon 10 and 30 minutes treatment with CaCl2 (200 mM) |
|
| Data table header descriptions |
| ID_REF |
|
| IA |
Raw Intensity value in channel A |
| IB |
Raw Intensity value in channel B |
| IA_NORM |
Lowess normalized value in channel A |
| IB_NORM |
Lowess normalized value in channel B |
| PRE_VALUE |
Ratio of Cy5/Cy3 label for each spot |
| VALUE |
same as UNF_VALUE but with flagged values removed |
| FlagA |
TIGR Spotfinder flag value in channel A; A - spot area is larger than 50 pixels; B - spot area is in the range between 30 and 50 pixels; C - spot area is smaller than 30 pixels; S - spot is fully or partialy saturated; U - spot is set by user for not to be processed; X - bad spot; Y - bad spot. Spot for which background is higher then signal; Z -spot is not detected |
| FlagB |
TIGR Spotfinder flag value in channel B; A - spot area is larger than 50 pixels; B - spot area is in the range between 30 and 50 pixels; C - spot area is smaller than 30 pixels; S - spot is fully or partialy saturated; U - spot is set by user for not to be processed; X - bad spot; Y - bad spot. Spot for which background is higher then signal; Z -spot is not detected |
| BkgA |
Background value in channel A |
| BkgB |
Background value in channel B |
| SDA |
Standard deviation for spot pixels in channel A |
| SDB |
Standard deviation for spot pixels in channel B |
| SDBkgA |
Standard deviation of the background value in channel A |
| SDBkgB |
Standard deviation of the background value in channel B |
| INV_VALUE |
1/PRE_VALUE, ie, (test/ref) ratio |
| UNF_VALUE |
log2 of INV_VALUE, ie, log2 (test/ref) ratio |
