Pathwayfinder rt2 profiler pcr array

The Human Signal Transduction PathwayFinder RT² Profiler PCR array (Qiagen, Hilden, Germany) was used to identify chemoresistance-linked pathways affected by treatment with BA and PEG–BA. This array contains crucial genes responsible for the activation or inhibition of several signalling processes involved in development, metabolism, immunology and stress-stimulation. It comprises five reference genes, one genomic DNA contamination control, three reverse transcription controls and three positive PCR controls. The sample mixture was prepared, and the assay performed according to the manufacturer’s instruction. A real-time PCR was conducted using the Bio-Rad CFX96 real-time touch detection system (Bio-Rad, Hercules, CA, USA). The CFX Maestro™ was used to generate Ct values and the Qiagen RT² PCR data analysis portal (https://geneglobe.qiagen.com/za/analyze/ (accessed on 12 May 2021)) used for differential gene expression analysis. The Qiagen tool was used to identify differentially expressed genes obtained by comparing PEG–BA-treated to BA-treated cells. This tool calculated fold change using the delta delta CT (2^−ΔΔCT) method [48 (link)].

The Human Signal Transduction PathwayFinder^™ RT2 Profiler^™ PCR Array (PAHS-014Z, Qiagen, Frederick, MD, USA) was used to evaluate the expression of a panel of 84 genes representative of ten different signal transduction pathways, in FaDu cells treated with pepsin. Total RNA was isolated from pepsin treated Fadu cells and control cells using the Qiagen RNeasy Mini Kit, following manufacturer’s protocol. RNA was quantified using the Nanodrop 2000 (Gene Company Limited, Hong Kong, China), and quality was assessed based on the integrity of 18 S and 28 S ribosomal RNA bands in 1% agarose gels. First-strand cDNA was mixed with 2 × RT2 SYBR Green qPCR Master Mix and ddH₂O. qPCR was performed in the Applied Biosystems (ABI) 7500 system using the following conditions: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each array contained five independent housekeeping genes (Actb, B2m, Hprt1, Ldha and Rplp1) that were used for data normalization.