Sybr primerscript real time pcr kit

According to the manufacturer’s protocol, total RNA was extracted using TRIzol reagent (Invitrogen), and the quality was assessed by spectrophotometric absorbance at 260/280 nm. First-strand cDNA was synthesized from 1 µg of total RNA using a cDNA synthesis kit (TaKaRa) following the manufacturer’s instructions. Reactions of quantitative real-time PCR (qPCR) were performed in a CFX96^TM Real-Time system (Bio-Rad, CA, U.S.A.) using the SYBR PrimerScript^TM real-time PCR kit (TaKaRa). The procedure included 1 cycle of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and primer-specific annealing temperature for 30 s. An 80-cycle melting curve was performed, starting at a temperature of 85°C and increasing by 0.5 every 10 s to determine primer specificity. Only one product of the desired size was identified and one single peak was observed in a melting curve for each primer. Each sample was repeated three times and the relative mRNA expression of genes was normalized to β-actin and GAPDH using the 2^{− ΔΔC}_t method [29 (link)]. The primers designed for qPCR are shown in Table 1.

For quantitative determination of the reliability of the sequencing data, qRT‒PCR was carried out to test the expression levels of 16 DEGs and 10 DEMs, which were randomly selected. Reverse transcription of mRNA to cDNA was performed using the RevertAid First Strand cDNA Synthesis Kit (K1622; Thermo Scientific) according to the manufacturer’s instructions. The relative mRNA expression level was determined using GAPDH as a housekeeping gene in each sample. Both forward and reverse primers are listed in Supplemental Table S1. U6 was chosen as the internal control for miRNA. Reverse transcription-PCR was used to synthesize cDNA using a miRNA 1st Strand cDNA Synthesis Kit (by stem‒loop) (Vazyme, Nanjing, China). Primers were obtained based on the mature sequences of the miRNAs identified in the present study (Supplemental Table S2). Mature sequences were obtained in the miRBase database, according to each miRNA's name. A CFX96TM real-time system (Bio-Rad, Hercules, CA, USA) was used to perform qRT-PCR with a SYBR PrimerScriptTM real-time PCR kit (TaKaRa, Dalian, China). The 2 − ΔΔCt method was used to measure the levels of relative expression, and expression differences were analysed by Student’s t test [82 (link)]. A P value of 0.05 was considered to indicate statistical significance.

Total RNA was extracted from the cultured cells using TRIzol (Invitrogen) at various time points (d1, d2, d3, d4, d5, d6, and d7) that corresponded to the MTT assay and image collection. First-strand cDNA was synthesized from 10 μg of total RNA using a cDNA synthesis kit following the manufacturer’s instructions (TaKaRa, Shiga, Japan). Levels of CYP17A1/19A1 were detected using the SYBR PrimerScript^TM real-time PCR kit (TaKaRa) and a CFX96^TM Real-Time system (Bio-Rad, CA, U.S.A.). The PCR was performed in a 25 μl reaction volume that consisted of 2.0 μl of cDNA, 12.5 μl of SYBR Premix EX Taq, 8.5 μl of sterile water, and 1.0 μl of each gene-specific primer. The raw results were repeated three times and normalized to β-Actin and GADPH using the 2^−ΔΔCt method [25 (link)]. Primers for these genes are listed in Table 1.