Geneup total rna mini kit

RNA was extracted from cultivated cells using the GeneUP total RNA mini Kit (Biotechrabbit; Henningsdorf, Germany), following the manufacturer’s recommendations, while respecting the stimulation protocol. The total RNA concentration and purity were evaluated photometrically. Total RNA was converted to cDNA using the QuantiTect reverse transcription kit (Qiagen; Hilden, Germany) following the manufacturer’s protocol. All primers were obtained from Biolegio (Nijmegen, The Netherlands) and were enlisted, along with the thermal cycler conditions (Table 1). Sample amplification and quantification were executed in the CFX Connect™ Real-Time PCR Detection System (Bio-Rad; Hercules, CA, USA). The integrity of the PCR products was evaluated by melting point analysis and agarose gel electrophoresis. Each sample was normalized to ribosomal protein L13a (RPL13a; ΔCq) as a reference gene and the experimental control condition (ΔΔCq). Mean fold changes were expressed as 2^(−ΔΔCq).

RNA from cultivated cells was isolated 24 h after treatment using the GeneUP total RNA mini Kit (Biotechrabbit, Henningsdorf, Germany), following the manufacturer’s protocol. Total RNA concentration and purity were evaluated photometrically. Total RNA (10 ng) was converted to cDNA by reverse transcription with the QuantiTect reverse transcription Kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. All primers used in this study were obtained from Biolegio (Nijmegen, The Netherlands). Primer sequences and PCR parameters are listed in Table 1. The samples were amplified and quantified on a Bio-Rad CFX Connect^TM real-time system (Hercules, CA, USA). PCR product integrity was evaluated by melting point analysis and agarose gel electrophoresis. The threshold cycle (CT) was normalized to ribosomal protein L13a (RPL13a; ΔCT). Data are depicted as 2^(−ΔΔCT). Real-time quantitative PCR (RT-qPCR) was performed in technical triplets, and each experiment was conducted in biological triplicate. Mean values +/− SEM were calculated for all samples.