This array includes tumor necrosis factor (TNF) ligands and their receptors, members of the bcl-2 gene family, caspases and some other important apoptosis-related genes. Briefly, total RNA was extracted from cell samples using an Array Grade Total RNA isolation kit (SuperArray, Frederick, MD) and quantitated by UV spectroscopy using a biophotometer. The integrity and quality of isolated RNA was determined by running the RNAs on agarose gel electrophoresis. Luminespib cDNA was labeled from total RNA with Biotin 16-dUTP and the GEArray® TM Amp Labeling-LPR Kit (SuperArray,
Frederick, MD) according to manufacturer’s instructions. The biotin-labeled cDNA was than added to the membrane and hybridized overnight to Human Apoptosis OligoGEArray® as stated
by the manufacturer. Signal detection was achieved by exposure to CDP-Star alkaline phosphatase chemiluminescent substrate (SuperArray, Frederick, MD). An image was processed using Kodak® Gel Logic 1500 Imaging System and analyzed with the GEArray Analyzer Software. Experiments were repeated thrice using RNA extracted from three different cultures. Real time quantitative RT-PCR (qRT-PCR) assay To validate our oligoarray results, quantitative real-time PCR was performed on four selected Combretastatin A4 chemical structure genes that were maximally effected by the combination treatment: lymphotoxin beta receptor (LTBR), myeloid cell leukemia-1 (MCL-1), tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), TNFRSF1A-associated death domain protein C59 research buy (TRADD). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positive control by using Real-Time™ qPCR Primer Assay (SABioscience, Frederick, MD) on Light Cycler 480 instrument (Roche Applied Science, Mannheim, Germany). Total RNA of 4 μg was extracted from cell samples using an
Array Grade Total RNA isolation kit (SuperArray, Frederick, MD) and quantitated by UV spectroscopy using a biophotometer. The integrity and quality of isolated RNA was determined by running the RNAs on agarose gel electrophoresis. PCR reaction mix was prepared 25 μl final volume containing 12,5 μl RT2 SYBR Green qPCR Master Mix, 10,5 μl DNAase-RNaseFree water, 1,0 μl gene-specific 10 μM PCR primer pair stock and finally 1,0 μl diluted cDNA samples for each primer (SABioscience). Universal cycling conditions (10 min at 95°C, 15 s at 95°C, 1 min 60°C for 40 cycles) were carried out. The melting protocol consisted of 95°C for 1 minutes and a continuous fluorescense reading from 65°C to 95°C at 30 acquisitions per degree and 1°C rising per second. Data normalization and analysis an endogenous control, GAPDH present on the PCR was used for normalization. Each replicate cycle threshold (CT) was normalized to the average CT of endogenous control on a sample basis. The comparative CT method was used to calculate the relative quantification of gene expression.