Cells were reverse transfected with 10nM control non targeting siRNA (dharmacon smart pool) or DUSP4-targeted siRNA. Some control-siRNA treated cells were also treated with 4 or 24 h of 1uM selumetinib prior to harvest. Remaining cells were treated with DMSO as a control for selumetinib. All cells were harvested 96h after transfection.
Growth protocol
MDA231 cells were grown in DMEM+10% FBS; BT549 cells were grown in RPMI + 10% FBS; SUM159PT cells were grown in DMEM/F12 nutrient mix + 5% FBS + 0.5ug/mL hydrocortisone
Extracted molecule
total RNA
Extraction protocol
RNEasy kit (QIAGEN)
Label
biotin
Label protocol
Affymetrix WT Sense reactions, using the Ambion WT Reaction kit (Catalog#4411974), were assembled and run following the manufacturer’s protocol. Briefly, a sample mix of 100ng total RNA, and poly-A spike in controls (included in Affymetrix WT Terminal Labeling Kit Catalog# 901524), were brought to a 5 ul final volume with nuclease free H2O. First Strand Synthesis Master mix was added to the sample mix and incubated at 25C for 1 hour, followed by incubation at 42 C for 1 hour. Incubations were followed by a cooling to 4 C for 2 minutes. Subsequently, Second Strand Synthesis Master mix was added to the sample mix, and incubated at 16C for 1 hours. The reaction was heatinactivated by 10 minute incubation at 65C. The resulting cDNA was then processed in a 16 hour IVT reaction to generate cRNA. The cRNA was then cleaned using magnetic beads included with the kit, and was used as template in a second first strand synthesis reaction to generate target of the correct sense for hybridization to the Affymetrix Gene and Exon arrays. The Second Cycle cDNA synthesis reaction was set up with 10ug cRNA, random hexamer, and first strand synthesis reagents to make cDNA. The cDNA target was then treated with Rnase H to remove template cRNA and cleaned up over a kit supplied column, or Agencourt SPRI beads, following manufacturer’s protocol (RNAClean, Catalog#000494). A total of 5.5ug of the clean cDNA target was then enzymatically fragmented and end-labeled using the Affymetrix kit reagents (Catalog# 901524). The cRNA, cDNA, and fragmented and end-labeled target were assessed by Agilent bioanalysis to insure that the amplified target met the recommended smear range, and that fragmentation and end-labeling were complete.
Hybridization protocol
For the Gene 1.1ST Array plate, the requisite amount of target was then added to hybridization cocktail for Gene Titan WT array plates as specified in the GeneChip® WT Terminal Labeling and Hybridization User Manual (P/N 702808 Rev. 2) to give a final target concentration of ~25ng/ul in the hybridization cocktail. The total amount of target hybridized was 2.25ug. The target in hybridization cocktail was heat denatured at 99C for 5 minutes, cooled to 45C for 5 minutes, centrifuged and then loaded on the Affymetrix Array plate arrays for hybridization on the Affymetrix Gene Titan Instrument.
Scan protocol
The Array plate was washed and stained on the Gene Titan instrument using Affymetrix Hybidization Wash and Stain kit reagents for the Gene Titan instrument (P/N 901406). After washing and staining, the array was scanned in the Affymetrix Gene Titan instrument using AGCC v.3.1.1
Description
BT549 cells transfected with DUSP4 siRNA for 96 h
Data processing
Signal was analyzed with Affymetrix Expression Console, v. 1.1, using an RMA normalization algorithm producing log base 2 results
