|
| Status |
Public on Jul 01, 2010 |
| Title |
AF293 from RPMI compared to AF293 with dendritic cells-9 h-rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
AF293 grown in RPMI for 9h
|
| Organism |
Aspergillus fumigatus |
| Characteristics |
strain: AF293
|
| Growth protocol |
Human immature dendritic cells (iDC), 6 x 105 ml-1, were co-incubated with A. fumigatus, 3 x 106 ml-1, in RPMI (10% FCS); MOI of 5. The samples were incubated at 37°C and 5% CO2 in 1.5 ml microcentrifuge tubes for 0, 3, 6, 9 and 12 hours; there were three replicates, each using dendritic cells from a different donor.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from A. fumigatus, with or without DC, immediately at each time point with the Ribo-Pure Yeast kit (Ambion).
|
| Label |
Cy3,Cy5
|
| Label protocol |
cDNA synthesis and labeling were performed as per JCVI PFGRC SOP m007 (http://pfgrc.jcvi.org/index.php/microarray/protocols.html) Briefly: cDNA were generated using Invitrogen Superscript II, incorporating aminoallyl-dUTP. cDNA Samples were labeled with Cy3 or Cy5 during a 2 hour room temperature incubation followed by purification with a Qiagen PCR purification column to remove unbound dye.
|
| |
|
| Channel 2 |
| Source name |
AF293 grown with dendritic cells in RPMI for 9h
|
| Organism |
Aspergillus fumigatus |
| Characteristics |
strain: AF293
|
| Growth protocol |
Human immature dendritic cells (iDC), 6 x 105 ml-1, were co-incubated with A. fumigatus, 3 x 106 ml-1, in RPMI (10% FCS); MOI of 5. The samples were incubated at 37°C and 5% CO2 in 1.5 ml microcentrifuge tubes for 0, 3, 6, 9 and 12 hours; there were three replicates, each using dendritic cells from a different donor.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from A. fumigatus, with or without DC, immediately at each time point with the Ribo-Pure Yeast kit (Ambion).
|
| Label |
Cy5,Cy3
|
| Label protocol |
cDNA synthesis and labeling were performed as per JCVI PFGRC SOP m007 (http://pfgrc.jcvi.org/index.php/microarray/protocols.html) Briefly: cDNA were generated using Invitrogen Superscript II, incorporating aminoallyl-dUTP. cDNA Samples were labeled with Cy3 or Cy5 during a 2 hour room temperature incubation followed by purification with a Qiagen PCR purification column to remove unbound dye.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed as per JCVI PFGRC SOP m008 (http://pfgrc.jcvi.org/index.php/microarray/protocols.html) Briefly: Hybridizations were incubated for 18 h at 42C. Post hybridization washes included (twice each) 55C in 2x ssc, 0.1% SDS, RT in 0.1x SSC, 0.1% SDS and RT 0.1x SSC.
|
| Scan protocol |
After washing, slides were scanned with a GenePix 4000b scanner using GenePix Pro 3.0 software. PMT gains were changed in order to generate images with red:green ratios of as close to 1:1 as possible. Images were saved as .TIF files and TIGR Spotfinder (Saeed et al, 2003) was used to quantitate fluorescent intensities. Poor quality spots were identified manually and flagged. After Spotfinder analysis, resulst were exported as TIGR software compatible MEV files following local background subtraction.
|
| Description |
Analysis of the response of A. fumigatus to human dendritic cells
|
| Data processing |
MEV files were loaded into the JCVI MIDAS array normalization program where they were first normalized by block locally-weighted scatter plot smoothing (LOWESS). After LOWESS, the flip-dye pairs were combined a consistency check was applied to remove any spots that do not fall within a specified deviation of the array-wide average log2 ratio of the fluorescence values for spot on both slides. After the flip-dye consistency check the values underwent inter-slide standard deviation regularisation by averaging the values of the replicate spots for each gene.
|
| |
|
| Submission date |
May 28, 2010 |
| Last update date |
Jun 01, 2010 |
| Contact name |
John Varga |
| E-mail(s) |
j.varga@yahoo.com
|
| Organization name |
Emory University
|
| Department |
Pediatrics
|
| Lab |
Goldberg
|
| Street address |
1510 Clifton Road
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30324 |
| Country |
USA |
| |
|
| Platform ID |
GPL10341 |
| Series (1) |
| GSE22053 |
The In Vitro Interaction of Aspergillus fumigatus and Human immature Dendritic Cells |
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