Bioarray rna labeling kit

Gene expression studies were performed as described [33 (link)]. Briefly, RNA was extracted and purified with the PureLinkTM Micro-to-Midi kit (Invitrogen, Paisley, UK). cDNA and biotinylated cRNA were synthesized using a T7-polyT primer and the BioArray RNA labeling kit (Enzo, NY, USA), respectively. Labeled RNA was hybridized to Human Gene 1.0 ST oligonucleotide arrays (Affymetrix, CA, USA). For the microarray data analysis, the .CEL files (in triplicate) were imported into the dChip software 19, and expression levels of the different PKC isoforms analysed. Levels of expression (arbitrary units) of the different PKC isoforms were plotted.

MDA-MB-231 cells were grown in DMEM with 10% of FBS and added to a final concentration of 500,000 cells/plate. Next day cells were treated with 500 nM EC-70124 for 24 h. Total RNA from 3 independent control samples and 3 independent treated cells was extracted and purified using the RNeasy Mini Kit (Qiagen). Double-stranded cDNA and biotinylated cRNA were synthesized using a T7 polyT primer and the BioArray RNA labeling kit (Enzo Life Sciences, Farmingdale, NY), respectively. The labeled RNA was fragmented and hybridized to human oligonucleotide arrays (Human Gene ST Arrays) (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Affymetrix CEL files were imported into the dChip software (Dana Farber Cancer Institute, Boston, MA): normalization of all arrays was done using a pair-wise rank invariant probe method and expression level of each probeset was calculated using the model-based expression index (MBEI).