|
| Status |
Public on Jan 24, 2008 |
| Title |
T98G 8 hr LY294002 vs Untreated Cy5-Cy3 rep 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Proliferating T98G cells with 8 hr LY294002 treatment
|
| Organism |
Homo sapiens |
| Characteristics |
T98G human glioblastoma cells
|
| Treatment protocol |
T98G human glioblastoma cells were grown in Minimal Essential Medium (Invitrogen) containing 10% fetal bovine serum (HyClone) and 100 units/ml of penicillin/streptomycin (Invitrogen). Cells were cultured for 48 hours, at which time they were actively proliferating with a doubling time of approximately 20 hours. If called for, LY294002 (Biomol) was added to a final concentration of 50 uM.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted with TRIzol reagent (Invitrogen) followed by poly(A)+ RNA isolation with an Oligotex mRNA Midi Kit (Qiagen) according to each manufacturer's protocol.
|
| Label |
Cy5
|
| Label protocol |
Starting with 100 ng of poly(A)+ RNA, one round of RNA amplification was performed with the MessageAmp aRNA Amplification Kit (Ambion) using a 4:1 amino-allyl UTP:UTP ratio for aRNA incorporation. For each sample, 8 micrograms of aRNA was coupled to N-hydroxysuccinimidyl esters of cyanine-3 or cyanine-5 (Amersham). Following clean-up, treated and untreated aRNA samples with opposing cyanine labels were combined, concentrated, and treated with a fragmentation reagent (Ambion) according to the manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
Proliferating T98G cells (untreated)
|
| Organism |
Homo sapiens |
| Characteristics |
T98G human glioblastoma cells
|
| Treatment protocol |
T98G human glioblastoma cells were grown in Minimal Essential Medium (Invitrogen) containing 10% fetal bovine serum (HyClone) and 100 units/ml of penicillin/streptomycin (Invitrogen). Cells were cultured for 48 hours, at which time they were actively proliferating with a doubling time of approximately 20 hours. If called for, LY294002 (Biomol) was added to a final concentration of 50 uM.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted with TRIzol reagent (Invitrogen) followed by poly(A)+ RNA isolation with an Oligotex mRNA Midi Kit (Qiagen) according to each manufacturer's protocol.
|
| Label |
Cy3
|
| Label protocol |
Starting with 100 ng of poly(A)+ RNA, one round of RNA amplification was performed with the MessageAmp aRNA Amplification Kit (Ambion) using a 4:1 amino-allyl UTP:UTP ratio for aRNA incorporation. For each sample, 8 micrograms of aRNA was coupled to N-hydroxysuccinimidyl esters of cyanine-3 or cyanine-5 (Amersham). Following clean-up, treated and untreated aRNA samples with opposing cyanine labels were combined, concentrated, and treated with a fragmentation reagent (Ambion) according to the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
For each slide, 4 micrograms of both treated and untreated cyanine-labeled aRNA samples were combined with a hybridization buffer (2.3X SSC, 18 mM HEPES, 0.2 mg/ml BSA, 0.6 mg/ml poly(A), 0.2% SDS), heat denatured for 3 minutes at 95 degrees C, and applied to microarrays under a LifterSlip coverslip (Erie Scientific). The slides were placed in a hybridization chamber (Dietech) and incubated in a 63 degree C water bath for 16 hr. Following hybridization, the slides were successively washed in 0.6X SSC with 0.025% SDS, 0.05X SSC, and water, then dried by centrifugation at 1000 rpm for 3 min.
|
| Scan protocol |
The microarrays were scanned with an Axon 4000B scanner and adaptive spot segmentation performed with GenePix Pro software (version 5.0) (Axon Instruments).
|
| Description |
For each treated sample, three independent replicate microarray experiments were performed.
|
| Data processing |
Triplicate dye-swap, background-subtracted median intensity values were used as input to the LIMMA analysis package (Smyth, 2005) in Bioconductor (Gentleman et al., 2004), and average LOESS-corrected log2 ratios were used to estimate differential gene expression after LY294002 treatment. Genes with a change in expression greater than or equal to 1.87 fold (log2 0.9) relative to untreated samples and false discovery rate (FDR)-corrected (Benjamini et al., 1995) moderated t-test p-values less than 0.01 were considered differentially expressed.
|
| |
|
| Submission date |
Jan 21, 2008 |
| Last update date |
Jan 23, 2008 |
| Contact name |
Geoffrey M Cooper |
| Organization name |
Boston University
|
| Department |
Biology
|
| Lab |
|
| Street address |
5 Cummington St
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL5444 |
| Series (1) |
| GSE10221 |
Inhibition of phosphatidylinositol 3-kinase signaling in proliferating human T98G cells |
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