|
| Status |
Public on Aug 31, 2007 |
| Title |
sglA- - 0mM cis - biological replication 2 - technical replication2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from sglA- cells, treated with 0mM cisplatin labeled with Alexa647
|
| Organism |
Dictyostelium discoideum |
| Characteristics |
sglA-
|
| Treatment protocol |
Exponentially growing cells (2x106 cells/ml) were divided into twelve 50 ml aliquots in 500 ml flasks. The cells were shaken for 30 min, 300 µM cisplatin was added to 6 cultures (PT buffer was added to the 6 controls), and the cells were shaken for 3 hr at 22C in the dark. Samples (1 ml) were drawn and assayed for viability. The remaining cells were washed with phosphate buffered saline (PBS) and resuspended in 1.0 ml TRIzol reagent (Life Technologies, Gaithersburg, MD) and RNA was extracted.
|
| Growth protocol |
Cells were grown in shaking suspension in HL5 medium, with the appropriate supplements for auxotrophy or drug selection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
2x106 cells/ml cells (treated or untreated) were collected, resuspended in 1 ml TRIZOL reagent (Life Technologies) and total RNA was extracted according to the manufacturer’s protocol. The RNA was resuspended in 20 mM MOPS (pH7.0) and its concentration was determined by spectrophotometry and verified by Northern blot analysis and staining with methylene blue, which also tested RNA integrity.
|
| Label |
Alexa647
|
| Label protocol |
Total RNA (10 µg) was mixed with 1 µg of labeled [dT(18)] primer in 13 µl of water, incubated at 70°C for 10 minutes and on ice for 2-5 minutes. Reaction buffer (Gibco-BRL), 0.1M DTT, 0.5 mM of each dNTP and 200U Superscript II (Gibco-BRL) were added and the reaction was incubated at 42°C for 2 hours. Experimental RNA samples were labeled with Cy5 primers and the reference RNA sample was labeled with Cy3. Both of the primers were HPLC-purified by the manufacturer (Operon Technologies). Reverse transcription reactions were terminated with 0.1M EDTA. RNA was degraded by adding 0.3 N NaOH and incubating at 60°C for 20 minutes. The reaction was neutralized with 0.4 M Tris.HCl pH7.6. Labeled cDNA was purified by ethanol precipitation, resuspended in 10 µl of DDW and mixed with 130 µl of PerfectHybTM Plus Hybridization buffer (SIGMA H-7033).
|
| |
|
| Channel 2 |
| Source name |
Total RNA from developed AX4 cells (0,3,6,12,17 and 24hours) labeled with Cyanine3
|
| Organism |
Dictyostelium discoideum |
| Characteristics |
strain: AX4
|
| Treatment protocol |
Exponentially growing cells (2x106 cells/ml) were divided into twelve 50 ml aliquots in 500 ml flasks. The cells were shaken for 30 min, 300 µM cisplatin was added to 6 cultures (PT buffer was added to the 6 controls), and the cells were shaken for 3 hr at 22C in the dark. Samples (1 ml) were drawn and assayed for viability. The remaining cells were washed with phosphate buffered saline (PBS) and resuspended in 1.0 ml TRIzol reagent (Life Technologies, Gaithersburg, MD) and RNA was extracted.
|
| Growth protocol |
Cells were grown in shaking suspension in HL5 medium, with the appropriate supplements for auxotrophy or drug selection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
2x106 cells/ml cells (treated or untreated) were collected, resuspended in 1 ml TRIZOL reagent (Life Technologies) and total RNA was extracted according to the manufacturer’s protocol. The RNA was resuspended in 20 mM MOPS (pH7.0) and its concentration was determined by spectrophotometry and verified by Northern blot analysis and staining with methylene blue, which also tested RNA integrity.
|
| Label |
Cyanine3
|
| Label protocol |
Total RNA (10 µg) was mixed with 1 µg of labeled [dT(18)] primer in 13 µl of water, incubated at 70°C for 10 minutes and on ice for 2-5 minutes. Reaction buffer (Gibco-BRL), 0.1M DTT, 0.5 mM of each dNTP and 200U Superscript II (Gibco-BRL) were added and the reaction was incubated at 42°C for 2 hours. Experimental RNA samples were labeled with Cy5 primers and the reference RNA sample was labeled with Cy3. Both of the primers were HPLC-purified by the manufacturer (Operon Technologies). Reverse transcription reactions were terminated with 0.1M EDTA. RNA was degraded by adding 0.3 N NaOH and incubating at 60°C for 20 minutes. The reaction was neutralized with 0.4 M Tris.HCl pH7.6. Labeled cDNA was purified by ethanol precipitation, resuspended in 10 µl of DDW and mixed with 130 µl of PerfectHybTM Plus Hybridization buffer (SIGMA H-7033).
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA in PerfectHybTM Plus Hybridization buffer (SIGMA H-7033) was hybridized to the arrays after heat treatment (95°C, 2 min) using a GeneTAC hybridization station (Genomic Solutions) according to the manufacturer’s recommended protocol. Labeled cDNA was hybridized to the arrays using a GeneTAC automatic hybridization station (Genomic Solutions) for 2 hours at 65C. Arrays were sequentially washed with 3 solutions, 2x SSC, 0.5% SDS; 0.5x SSC, 0.5% SDS; 0.1x SSC, for 30 sec at room temperature twice each.
|
| Scan protocol |
The arrays were scanned with a ScanArray5000 scanner (GSI Lumonics) according to the manufacturer’s recommended protocol. PMT and Laser settings were not recorded.
|
| Description |
NA
|
| Data processing |
All images were processed with the Imagene software package.
|
| |
|
| Submission date |
Jun 25, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Nancy E Van Driessche |
| Organization name |
Baylor college of medicine
|
| Lab |
Shaulsky lab
|
| Street address |
One Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77006 |
| Country |
USA |
| |
|
| Platform ID |
GPL5382 |
| Series (1) |
| GSE8287 |
Global Transcriptional Responses to Cisplatin in Dictyostelium discoideum Identify Potential Drug Targets |
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