Quantitect transcription kit

RNA from cultivated cells was isolated using the GeneUP total RNA mini Kit (Biotechrabbit, Henningsdorf, Germany) according to the manufacturer's protocol. RNA concentration and purity were evaluated photometrically (FLUOstar Omega, BMG LAB-TECH, Ortenberg, Germany). According to the manufacturer's recommendations, the total RNA (10 ng) was converted to cDNA by reverse transcription with the QuantiTect Transcription Kit (Qiagen, Hilden, Germany). 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 LightCycler 96 (Roche, Mannheim, Germany). PCR product integrity was evaluated by melting point analysis and agarose gel electrophoresis. The threshold cycle (CT) was normalized to housekeeping ribosomal protein L13a (RPL13a; ΔCT). Data are depicted as 2 (-ΔΔCT) . RT-qPCR was performed in technical triplets, and each experiment was conducted in at least biological triplicate. Mean values ± SEM were calculated for all samples.

To evaluate the expression of porcine JAZF1 in different tissues, RNA was isolated from 10 mg tissue using the RNA extraction kit (Qiagen, China) following manufacturer instructions. Total cDNA was generated with QuantiTect Transcription Kit (Qiagen). SYBR Green primers were designed using the Premier 5.0 software (Table 1). qPCR was carried out in a real-time PCR System (ABI, USA). The results were calculated with the relative quantification method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. JAZF1 expression levels were calculated by the 2 -∆∆Ct comparative method. qPCR amplifications were carried out in a final volume of 20 µL comprising 10 µL 2X qPCR Mix (ABI), 1 µL of each primer (10 µM), 1 µL template cDNA, and 8 µL ddH 2 O with the following program: 95°C for 2 min and 40 cycles of 95°C for 10 s, 60°C for 40 s, and 72°C for 30 s.