|
| Status |
Public on Jul 28, 2008 |
| Title |
2h Phagocytosis by RAW Cells Replicate 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
No macrophage control
|
| Organism |
Candida albicans |
| Characteristics |
Time: 2h
|
| Treatment protocol |
No macrophage control
|
| Growth protocol |
Candida cells were grown overnight at 30°C in yeast extract-peptone-dextrose medium (YPD), washed twice in PBS, counted with a hemacymeter and resuspended just before the interaction at 10e8 cells / 30 ml of warm DMEM supplemented with 10% FBS (D-10). The macrophage cell lines were maintained in D-10 medium. For the interaction assays, 150 mm or 60 mm Petri dishes were seeded 24 hours before with 33X10e7/30 ml or 14X10e7/10 ml of macrophage cells respectively and medium was replaced with 30 ml or 10 ml respectively of a Candida suspension to give a final MOI of 1.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
1 to 4 X 150 mm dishes were harvested by a quick wash in D-PBS, followed by addition of 10 ml/plate (Candida only) or 15 ml/plate (Candida with macrophages) of Trizol reagent. Cells were collected and centrifuged at 12000 X g. Pellets contained intact Candida cells, and the pellet containing Candida with macrophages was washed 2 more times with Trizol to remove contaminating macrophage DNA and RNA. Intact Candida cell pellets were quickly frozen at –80°C. Total RNA was isolated using the hot phenol extraction protocol repeated three times (Kohrer, K., and H. Domdey. 1991. Preparation of high molecular weight RNA. Methods Enzymol 194:398-405). Total RNA was further purified using RNeasy mini kit (Qiagen) according to the manufacturer instructions. RNA quantification was assessed by absorbance reading at 260 nm (Nanodrop) and its quality was assessed using an Agilent 2100 Bioanalyzer (Santa Clara, CA).
|
| Label |
Cy5
|
| Label protocol |
7.5 to 20 µg of total RNA was reverse transcribed using oligo(dT)20 in the presence of Cy5-dCTP or Cy3-dCTP (Amersham) and Superscript III reverse transcriptase (Invitrogen) . Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 30 min at 37°C. The labeled cDNAs were purified with Illustra Cyscribe GFX purification kit (GE Healthcare).
|
| |
|
| Channel 2 |
| Source name |
Co-cultured with RAW264.7
|
| Organism |
Candida albicans |
| Characteristics |
Time: 2h
|
| Treatment protocol |
Co-cultured with RAW264.7
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
1 to 4 X 150 mm dishes were harvested by a quick wash in D-PBS, followed by addition of 10 ml/plate (Candida only) or 15 ml/plate (Candida with macrophages) of Trizol reagent. Cells were collected and centrifuged at 12000 X g. Pellets contained intact Candida cells, and the pellet containing Candida with macrophages was washed 2 more times with Trizol to remove contaminating macrophage DNA and RNA. Intact Candida cell pellets were quickly frozen at –80°C. Total RNA was isolated using the hot phenol extraction protocol repeated three times (Kohrer, K., and H. Domdey. 1991. Preparation of high molecular weight RNA. Methods Enzymol 194:398-405). Total RNA was further purified using RNeasy mini kit (Qiagen) according to the manufacturer instructions. RNA quantification was assessed by absorbance reading at 260 nm (Nanodrop) and its quality was assessed using an Agilent 2100 Bioanalyzer (Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
7.5 to 20 µg of total RNA was reverse transcribed using oligo(dT)20 in the presence of Cy5-dCTP or Cy3-dCTP (Amersham) and Superscript III reverse transcriptase (Invitrogen) . Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 30 min at 37°C. The labeled cDNAs were purified with Illustra Cyscribe GFX purification kit (GE Healthcare).
|
| |
|
| |
| Hybridization protocol |
The microarray slides were diped sequentially in 0.2% SDS and water and immediately pre-hybridized for 1 hour at 42°C with 5X SSC, 0.1% SDS and 0.1% BSA and subsequently washed twice for 5 min with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer (Roche) with yeast tRNA (Invitrogen) and salmon sperm DNA (Sigma) in a SlideBooster hybridization chamber (Advalytix). The slides were washed twice with SSC-0.1% SDS, once with 1X SSC and once with 0.1X SSC. Slides were dried by centrifugation.
|
| Scan protocol |
Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 10-µm resolution.
|
| Description |
RAW-2h-rep4
|
| Data processing |
Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).
|
| |
|
| Submission date |
May 09, 2008 |
| Last update date |
Jul 28, 2008 |
| Contact name |
Andre Nantel |
| E-mail(s) |
andre.nantel@nrc-cnrc.gc.ca
|
| Phone |
514-496-6370
|
| Fax |
514-496-9127
|
| Organization name |
National Research Council of Canada
|
| Department |
Biotechnology Research Institute
|
| Street address |
6100 Royalmount
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H4P 2R2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6822 |
| Series (1) |
| GSE11399 |
Transcriptional Response of Candida albicans Cells Following Phagocytosis by Mouse Macrophages |
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