|
| Status |
Public on Jun 13, 2008 |
| Title |
10um Anacardic acid treated for 12 h Replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
AA treated parasites
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: 3D7
|
| Treatment protocol |
3*10^9 18 h old synchronized ring were treated for 12 h with 0, 5, 10, or 20 μM of Anacardic acid
|
| Growth protocol |
Parasites were cultured in human O+ erythrocytes at 5% hematocrit in RPMI 1640 supplemented with 25 mM Hepes, pH 7.5, 25 mM sodium bicarbonate, 50 mg/L hypoxanthine, 0.5% Albumax II, and 40 µg/ml gentamicin sulfate. Cultures were maintained at 37oC in a gas mixture of 5% CO2 and 3% O2. Synchronization was carried out by two 5% sorbitol treatments of ring-stage parasites
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from control and treated parasites using TRIzol Reagent and treated with RNase free DNase I (Promega, Madison, WI) to remove contaminating genomic DNA.
|
| Label |
Cy5
|
| Label protocol |
Fluorescence labeling with dUTP-Cy3 or dUTP-Cy5 was performed by reverse transcription using 30 µg of total RNA with both oligo(dT)12-20 and random hexamers according to the manufacturer’s instruction (Amersham).
|
| |
|
| Channel 2 |
| Source name |
AA untreated control
|
| Organism |
Plasmodium falciparum |
| Characteristics |
strain: 3D7
|
| Treatment protocol |
3*10^9 18 h old synchronized ring were treated for 12 h with 0, 5, 10, or 20 μM of Anacardic acid
|
| Growth protocol |
Parasites were cultured in human O+ erythrocytes at 5% hematocrit in RPMI 1640 supplemented with 25 mM Hepes, pH 7.5, 25 mM sodium bicarbonate, 50 mg/L hypoxanthine, 0.5% Albumax II, and 40 µg/ml gentamicin sulfate. Cultures were maintained at 37oC in a gas mixture of 5% CO2 and 3% O2. Synchronization was carried out by two 5% sorbitol treatments of ring-stage parasites
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from control and treated parasites using TRIzol Reagent and treated with RNase free DNase I (Promega, Madison, WI) to remove contaminating genomic DNA.
|
| Label |
Cy3
|
| Label protocol |
Fluorescence labeling with dUTP-Cy3 or dUTP-Cy5 was performed by reverse transcription using 30 µg of total RNA with both oligo(dT)12-20 and random hexamers according to the manufacturer’s instruction (Amersham).
|
| |
|
| |
| Hybridization protocol |
To 28 microliter buffer (30 mM sodium citrate, 300 mM sodium chloride, 50% formamide, 0.4% SDS, pH 7.0) at 42 deg. C.,was added 25 microliter of each sample of labeled cDNA along with 3 microliter of 1:1 salmon sperm/tRNA blocker (concentration?). After heating for 2 min at 98 deg. C, centrifugation for 3 min at 14,000 x g, 56 microliter of supernatant was transferred into a microarray hybridization chamber (MAUI FL Mixer, Biomicro Systems). After overnight hybridization at 45 deg. C, chambers were inserted into a jig and submerged in Buffer A, comprised of 1X SSC and 0.05% SDS. The MAUI Mixer lid was removed and the microarray slides were placed in a rack submerged in fresh Buffer A. Once all the slides were in the rack, they were washed three times in fresh buffer A, shaking 2 min for each wash. Then the slides were transferred and washed three times in fresh 0.1X SSC buffer, shaking 2 min per wash followed by centrifugation for 5 min at 580 RPM. Once dry, the slides were removed from the centrifuge and scanned on a GenePix 4000B Scanner (Axon Instruments/Molecular Devices).
|
| Scan protocol |
Scanned on a GenePix 4000B scanner.
|
| Description |
Same amount of labeled cDNA from treated parasites or untreated control was mix together
Pfab-11-18
mAdb ID: 84857
|
| Data processing |
Images were quantified using GenePix Pro 6. Signal calculation: Median Intensity minus Median Background. Normalization: 50th percentile using spot filtered genes. Spot Filter Options: Include spots not flagged bad or not found and both Chan A and Chan B signal >= 2 SD above background. Within array multiple occurrences of Well ID were reduced to a single instance by selecting the one with the strongest signal.
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| |
|
| Submission date |
Jun 12, 2008 |
| Last update date |
Jun 12, 2008 |
| Contact name |
Tetsuya Furuya |
| E-mail(s) |
tfuruya@niaid.nih.gov
|
| Phone |
301-402-7612
|
| Organization name |
National Institutes of Health
|
| Street address |
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20892-8130 |
| Country |
USA |
| |
|
| Platform ID |
GPL1858 |
| Series (1) |
| GSE11763 |
The HAT Inhibitor Anacardic Acid Leads to Changes in Global Gene Expression During in vitro P.falciparum Development |
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