Human apoptosis rt2 profiler pcr array

After incubation in the absence or presence of TNF‐α (10 ng/mL), neutrophils were pelleted, and RNA from the pellet was extracted with Trizol reagent using an RNAeasy kit, which included a DNA digestion step. Total RNA concentration and integrity was assessed using the Agilent 2100 Bioanalyser RNA Nano chip. cDNA was synthesized from 200 ng of total RNA from each sample, using the Superscript III First Strand cDNA Synthesis kit, in a 20 μL reaction volume, as per the manufacturer's instructions. Quantitative PCR analysis was carried out with the SYBR Green PCR kit, as per the manufacturer's instructions, using a Roche 480 LightCycler in a 96‐well plate using 1 μL of the above cDNA suspension in a 20 µL reaction volume. Target gene expression was quantified against a panel of housekeeping genes (GAPDH, β‐2 microglobulin, β‐actin, peptidylprolyl isomerase A). Initially, a Human Apoptosis RT² Profiler PCR Array (SA Biosciences) was used to detect neutrophil transcripts whose levels were regulated by TNF‐α treatment. Thereafter, specific primers for genes of interest were purchased from PrimerDesign and Eurofins (Table 1). Levels of gene expression were normalized to the housekeeping genes by comparing Ct values, and data presented are normalized to GAPDH 30.

Total RNA was extracted from DU145, PWR-1E and RWPE-1 cells using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Extracted RNA was reverse-transcribed into complementary DNA (cDNA) using iScript cDNA Synthesis kit (Bio-Rad) and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR analysis was performed with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan Universal PCR master mix according to the manufacturer's protocol (Applied Biosystems). Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems). PCR parameters for cycling were as follows: 95ºC for 20 seconds, then 40 cycles of 95ºC for 3 seconds and 60ºC for 30 seconds. All reactions were done in a 10 μL reaction volume in triplicate. The data were analyzed using the delta-delta Ct method to calculate the fold-change. TaqMan probes and primers for PMS2 (assay ID: Hs00241053_m1), TMS1 (Hs01547324_gH), BCL2A1 (Hs00187845_m1), and GAPDH (Hs02758991_g1) were obtained from Applied Biosystems. GAPDH was used as internal control. For array analyses, expression of apoptosis-related genes was determined using the Human Apoptosis RT2 Profiler PCR Array (Qiagen) per manufacturer's instructions.

2 × 10⁶ SW620 and HCT116 (parental and OxR) cells were seeded into a 100-mm-diameter cell culture dish for 24 hr. Cells were lifted using a cell scraper and washed with HBSS with calcium and magnesium. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA yield following isolation was determined using a UV5Nano spectrophotometer (Mettler Toledo). cDNA synthesis was completed using the RT² First Strand Kit (Qiagen, 330404) using 0.5 μg RNA per sample. RNA expression of 84 apoptotic genes was analyzed using the RT2 Profiler PCR Human Apoptosis Array (Qiagen, PAHS-012Z). Arrays were prepared according to the manufacturer’s protocols applied to the prepared cDNA samples. Profiler array plates were run on a CFX96 Touch Real Time PCR (Bio-Rad) using the following protocol: 1 cycle for 10 min at 95°C, 40 cycles of 95°C for 15 s followed by 60°C for 60 s at a rate of 1°C/s. Melt curves were generated immediately following the PCR protocol. Cycle threshold (Ct) values were calculated using CFX Maestro Software (Bio-Rad). Data analysis was completed using the GeneGlobe Data Analysis Center (Qiagen). Volcano plots were generated in GraphPad Prism using calculated fold changes in gene expression between OxR and parental cells and their corresponding p-values.