All in one rt supermix perfect for qpcr kit

For gene expression analysis, cDNA synthesis was performed using an All-in-one RT SuperMix Perfect for qPCR kit (Vazyme, NanKing, China) according to the manufacturer’s instructions. qPCR was used to verify the levels of expressed genes, with glyceraldehyde-3-phosphate (GAPDH) used as the internal reference gene. Vazyme ChamQ Universal SYBR qPCR Master Mix Q711-02 (Vazyme, NanKing, China) and Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA) were used for qPCR. Information on the qPCR primers is shown in Supplementary File S2.

The cDNA synthesis from total RNA was performed with All-in-One RT SuperMix Perfect for qPCR kit (Vazyme). A total of 20 μL reaction system consisted of 10 μL PowerUP™ SYBR™ Green Master Mix (ABI), 2 μL primer mixture, 7 μL sterile water, and 1 μL cDNA template. The qRT-PCR was performed on the QuantStudiO™ 3 Real-Time PCR Instrument (96-Well Block, ABI) with the following protocol: initial denaturation for 2 min at 50°C, 2 min at 95°C, 50 cycles for 15 s at 95°C, 30 s at 58°C, followed by a further 15 s at 95°C, 1 min at 60°C, 1 s at 95°C. The β-actin was used as the internal reference gene to calculate the folding change of expression level of target genes using the 2^(-ΔΔCt) method. The specific primers are listed in Supplementary Table 1.

The total RNA of C. elegans samples was isolated using the RaPure Total RNA Kit (Magen, Shanghai, China) following the manufacturer’s protocol. In brief, the RLT lysis buffer was used to lyse the worms, and then total RNA was collected and purified using HiPure RNA Mini Columns. Finally, the RNA was dissolved with RNase free water. The purity and concentration of RNA were determined using a NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA). cDNA was synthesized using the HiScript III. All-in-one RT SuperMix Perfect for qPCR Kit (Vazyme, Nanjing, China). Relative quantitative RT-PCR was performed using Rotor-Gene Q (Qiagen, Beijing, China) with a Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China). RT-PCR amplification conditions included denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, and extension for 32 s at 72 °C. Act-1 was used for normalization. All primer pairs were synthesized by Synbio Technologies (Jiangsu, China), and the sizes of PCR products were between 100 to 300 bp. The primer sequences used are listed in Table 6. Results were analyzed using the comparative threshold cycle (Ct) method and expressed as the fold change in gene expression (2^−ΔΔCt).