Rt2 profiler pcr array system kit

After compound exposure, RNA was extracted from the cell lines using TRIzol^® (Invitrogen, Carlsbad, CA, USA), according to manufacturer’s instructions. The extracted RNA was quantified using a Nanodrop Life Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Furthermore, 400 ng of complementary DNA (cDNA) was synthetized using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland), according to manufacturer’s instructions. The RT² Profiler PCR Array System Kit (Qiagen, Hilden, Germany) included 96 genes corresponding to cancer research molecular pathways and adequate controls in quadruplicate. The expression levels were determined by real-time PCR in a LightCycler 480 (Roche Diagnostics) and ACTINB, GAPDH, and HPRT1 were used as endogenous controls. The RT² profiler PCR array analysis was performed using the Qiagen-specific platform. The data analysis in the web portal calculated fold change using the ΔΔCT method. Genes with a logarithmized fold change above 1 or below −1 were considered. Additionally, the DNA genomic contamination (GDC), as well as the first strand synthesis (RTC) and real-time PCR efficiency (PPC), were monitored using the Qiagen platform for the RT² profiler PCR array analysis. The lower limit of detection was set at CT ≥ 35.

Total RNA was extracted from NCCIT cell line (four biological replicates) using TRIzol (Invitrogen, Carlsbad, CA, USA) and quantified in NanoDropTM Lite Spectophotometer (Cat. ND-LITE, Thermo ScientificTM, Waltham, MA, USA). Four hundred nanograms of cDNA were synthetized using Transcriptor High Fidelity cDNA Synthesis Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. The RT² Profiler PCR Array System Kit (Qiagen, Hilden, Germany) included 96 genes corresponding to cancer research molecular pathways and adequate controls, in quadruplicates. The expression levels were determined by real-time quantitative PCR in a LightCycler 48 platform (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and ACTINβ, β2M, GAPDH, HPRT1, and RPLP0 were used as endogenous controls. The RT² profiler PCR array analysis was performed in Qiagen specific platform. The data analysis in web portal calculates fold change using ΔΔCt method. Genes with a logarithm fold change above 1.5 or below −1.5 were considered. Additionally, DNA genomic contamination, as well as first strand synthesis and real-time PCR efficiency were monitored in Qiagen platform for RT² profiler PCR array analysis. The lower limit detection was set at Ct ≥ 35.