Mirna universal sybr qpcr master mix kit

Two types of samples were used for quantitative PCR (qPCR). One was the embryos/larvae at the different stages (18, 21, 25, 42 hpf and 2, 4 dpa), another was the wild-type and miR4018a/4000f/4018b-overexpressed larvae collected at 31 hpf. The total RNA was extracted with RNAiso Reagent (Takara). All cDNAs were synthesized using the miRNA First Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China). qPCR amplification was performed using the miRNA Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) on the Roche Applied Science LightCycler^TM 480 Real-Time PCR System. According to the supplier’s protocol, the reaction was carried out with the following conditions: 95°C for 5 min, 40 cycles of 95°C for 10 s and 60°C for 30 s, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. Data were calculated using the 2^−ΔΔCt method, and statistical analyses were performed using paired Student’s t-tests. A value of p < 0.05 was considered statistically significant.

TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA) was used to obtain total RNA according to the manufacturer's instructions. The cDNA was synthesized and amplified using a miRNA 1st Strand cDNA Synthesis Kit and miRNA Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) according to the manufacturer's protocols. The sequences of primers are listed in Table S1. The relative levels of target genes were normalized to GAPDH by 2^ΔΔCt method.

A 50 µL reaction mixture, which contained 5 µL of the cDNA template, 2 µL each (10 µM) of forward and reverse primers, 25 µL of 2 × miRNA Universal SYBR qPCR Master Mix, and 16 µL of ddH₂O, was prepared according to the instructions of the Vazyme miRNA Universal SYBR qPCR Master Mix Kit. Each reaction was performed in triplicate. The PCR was conducted using the following program: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, followed by annealing at 60 °C for 30 s, and a final extension step at 72 °C for 15 s. A melting curve analysis was conducted using the following temperature program: 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. Each reaction system was run in triplicate.
The amplification efficiency of the primers was determined by generating a standard curve using a fivefold dilution series of the cDNA template in water (v:v) –at 1:4, 1:24, 1:124, 1:624, and 1:3124. After each dilution, qRT-PCR was performed for each primer pair to obtain the Cq values and generate the standard curve. The amplification efficiency (E) and the R² value were calculated, and the correction equation was determined to be: E = (5 ^−1/slope − 1) × 100%^{27 ,28} .

RNA extracting solution (Servicebio, China) was used to extract total RNA from transfected cells. Quantitative real-time PCR (qRT-PCR) was performed according to Taq Pro Universal SYBR qPCR Master Mix kit (Vazyme, China) and miRNA Universal SYBR qPCR Master Mix kit (Vazyme, China). Relative sequences of the primers were listed in Table S1. β-actin or U6 small nuclear RNA was applied as internal control.