Primescript rttm reagent kit

The total RNA was extracted from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions, then the RNA quality, purity, and concentration were measured by spectrophotometric absorbance measurement. The cDNA was further synthesized from 1 μg of total RNA using a PrimeScript RT^TM Reagent Kit (TaKaRa, Dalian, China), in accordance with the manufacturer’s instructions. The qRT-PCR analysis was conducted using 2 × SYBR Premix Ex Taq II (TaKaRa, Dalian, China). The reaction solution was prepared in a total volume of 12.5 μl containing 1 μl cDNA, 6.25 μl of 2 × SYBR Premix Ex Taq, 4.25 μl of ddH₂O, and 0.5 μl of each gene-specific primer (10 μM). For each sample, the analysis was conducted in triplicate and normalized to β-actin by the 2^–ΔΔCt method (Livak and Schmittgen, 2000 (link)). The control was set as one. The primers for qRT-PCR are summarized in Table 2.

Following the TRIzol reagent (Invitrogen, Carlsbad, CA, United States) extraction of total RNA, spectrophotometric absorbance measurements were conducted to determine the quality, purity, and concentration of RNA. According to the manufacturer’s instructions, the cDNA was further synthesized from total RNA using a PrimeScript RTTM Reagent Kit (TaKaRa, Dalian, China). The 2 × SYBR Premix Ex Taq II (TaKaRa, Dalian, China) was employed to conduct the qRT-PCR. Using 1 μL cDNA, 6.25 μL of SYBR Ex Taq, 4.25 μL of ddH₂O, and 0.5 μL of each gene-specific primer (10 μM), a total of 12.5 μL reaction solution was prepared. For each sample, the Ct value of each gene were normalized to GAPDH using the 2^–ΔΔCt method [43 (link)] and significance analyses were repeated three times through t-test, one–way ANOVA, and non-parametric test. The primers for qRT–PCR are summarized in Table 1.