Gdna digester plus

Total RNA was extracted from the hypothalamus tissues using Molpure^® TissueTotal RNA Kit (YEASEN Biotechnology (Shanghai) Co., Ltd.). In addition, the cDNA was synthesized using a gDNA digester plus (YEASEN Biotech Co., Ltd.). Then, the Hieff UNICON^® Universal Blue SYBR Green Master Mix (YEASEN Biotech Co., Ltd.) was used to execute an amplification reaction in Gene-9660 System (Bioer Technology, Hangzhou, China). The amplification conditions were 95°C for 5 min, 40 cycles at 95°C for 10 s, and 60°C for 30 s. Relative fold change was calculated using the −2ΔΔCT method using Gapdh as the control marker. Primer sequences are demonstrated in Supplementary Table 1.

RT PCR for AKT3 transcripts was performed as previously reported [15 (link)]. Total RNA (2 μg) extracted from the hippocampi of aged mice was reverse transcribed to cDNA using the Hifair® III 1st strand cDNA synthesis kit containing gDNA digester plus (Yeasen, China). The hippocampi of mice were injected with the recombinant adeno-associated virus to overexpress circAKT3 (rAAV-hSyn-mmu-circAKT3-nEF1α-EGFP) or the empty vector (rAAV-hSyn-EGFP) to exclude the interference of rAAV injections. Next, cDNA was amplified using 2 × Phanta^® Max Master Mix with Dye Plus (Vazyme, China) and common sense primer (5′-GGACTATCTACATTCCGGAAAG-3′) with AKT3 transcript 1 primer (5′-GGTGAAGACCCTTGGCTGGTC-3′) or AKT3 transcript 2 primer (5′-GGGTCTAGATTACTTTTTATTATCATTTTTTTTCCAGTTAC-3′). The primer sequences were synthesized as reported previously [15 (link)]. And the PCR protocol consisted of preliminary denaturation (95 ℃, 3 min), repeated 35 cycles containing denaturation (95 ℃, 15 s), annealing (54 ℃, 15 s) and extension (72 ℃, 30 s), and a further extension at 72 ℃ for 5 min. Afterwards, the Semiquantitative RT-PCR was scored by agarose gel electrophoresis (1%) mixed with GelRed nucleic acid dye (Yeasen, China) and photographed by the ultraviolet imaging system.