cell line: 5637 cell type: Bladder cancer cell line treatment: DMSO agent: GSK126
Treatment protocol
Bladder cancer cell lines were seeded in 6-well plates. After 12 h overnight incubation, the cells were treated either with DMSO or 7.5 uM of GSK126.
Growth protocol
Bladder cancer cell lines HT1197 (ATCC, Manassas, VA, USA), HT1376 (ATCC), T24 (ATCC), 5637 (ATCC) were grown in RPMI-1640 (Life Technologies, Carlsbad, CA, USA) with penicillin–streptomycin (100 U/ml) and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in 5% CO2 cell culture incubator.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from bladder cancer cell lines using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA).
Label
Cy3
Label protocol
1 µg of total RNA were primed with 2 µl of 100 µM T7-oligo-dT DNA primer at 65°C for 10 min, then reversed transcribed at 40°C for 2 h in the presence of 400 U MMLV RTase (Agilent), and labeled with T7 polymerase with 100 µM each of dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-label or Cy3-label.
cell line: 5637 cell type: bladder cancer cell line
Treatment protocol
Bladder cancer cell lines were seeded in 6-well plates. After 12 h overnight incubation, the cells were treated either with DMSO or 7.5 uM of GSK126.
Growth protocol
Bladder cancer cell lines HT1197 (ATCC, Manassas, VA, USA), HT1376 (ATCC), T24 (ATCC), 5637 (ATCC) were grown in RPMI-1640 (Life Technologies, Carlsbad, CA, USA) with penicillin–streptomycin (100 U/ml) and 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in 5% CO2 cell culture incubator.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from bladder cancer cell lines using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA).
Label
Cy5
Label protocol
1 µg of total RNA were primed with 2 µl of 100 µM T7-oligo-dT DNA primer at 65°C for 10 min, then reversed transcribed at 40°C for 2 h in the presence of 400 U MMLV RTase (Agilent), and labeled with T7 polymerase with 100 µM each of dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-label or Cy3-label.
Hybridization protocol
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially.
Scan protocol
Scanned on an Agilent G2505B scanner. Images were quantified using Agilent Feature Extraction Software.
Description
5637_DMSO Vs GSK126
Data processing
Data from the two channel experiment arrays were lowess normalized usinf 'agilp' bioconductor package. Then, differential expression is measured by log2(reference/test).
