Viia 7 dx system

Total RNA was extracted using RNAiso Plus reagent (Takara, Dalian, Liaoning, China). Reverse transcription (1 μg of total RNA) was performed with the PrimeScript RT Reagent Kit (code No. RR047A; Takara, Dalian, Liaoning, China). The cDNA was subjected to qPCR on a ViiA^TM 7 Dx system (Applied Biosystems, Foster, CA, United States) using SYBR Green qPCR Master Mix (Takara, Dalian, Liaoning, China). The expression levels of the target genes were normalized to the expression of an internal control gene (rpoD) using the 2^–ΔΔCt method. The sequences of the primers are listed in Supplementary Table S2.

A ViiA^TM 7 Dx system (Applied Biosystems) was used as a platform to
quantify gene transcripts by real-time PCR. We analyzed four samples C.
macropomum liver for each treatment. The reaction mixture consisted of 1
μL of cDNA as template (added in triplicate to the wells of a 96-well thin-wall PCR
plate), 1 μL of each primer (concentration of ras: 2 pmol;
hif-1α: 2 pmol, 28S: 2.5 pmol and
ef-1α: 1.5 pmol), 2 μL of nuclease-free water 192 (Ambion, Life
Technologies) and 5 μL of SYBR Green PCR Master Mix (Applied Biosystems) in a total
volume of 10 μL. The PCR protocol was: 2 min at 50 °C and 95 °C for 10 min, followed
by 40 cycles of 95 °C for 15 s and 60 °C for 1 min (annealing temperature of all
primers). By melting curve analysis the presence of a single product-specific melting
temperature was confirmed: 28S (slope -3.36/ R² 0.99),
ef-1α (slope -.3.34/ R² 0.99), ras(slope -3.33/ R² 0.97) and hif-1α (slope -3.20/
R² 0.99). Amplification efficiency for each primer set was calculated
from a serial dilution curve obtained from a pool of experimental samples (1000 to 1
ng cDNA concentration; n = 4). All primer pairs showed high PCR efficiency (between
98–105%). Serial dilutions of a cDNA standard were amplified in each run to determine
amplification efficiency according to Pfaffl
(2001).

Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian, Liaoning, China). Reverse transcription (1 μg of total RNA) was performed with the PrimeScript RT reagent kit (TaKaRa, Dalian, Liaoning, China). The cDNA was subjected to quantitative PCR (qPCR) on a ViiA 7 Dx system (Applied Biosystems, Foster, CA, United States) using SYBR green qPCR master mixes (TaKaRa, Dalian, Liaoning, China). The expression levels of target genes were normalized to that of the internal control gene (rpoD) using the 2^–ΔΔCt method. More details about primers are listed in Supplementary Table 3.

Total RNA was extracted using total RNA isolation reagent (Promega, Madison, WI, USA). Reverse transcription (1 μg of total RNA) was performed with the PrimeScript RT reagent Kit (Takara, Dalian, Liaoning, China). The cDNA was subjected to qPCR on a ViiA™ 7 Dx system (Applied Biosystems, Foster, CA, USA) using SYBR Green qPCR Master Mixes (ThermoFisher Scientific). The expression levels of the target genes were normalized to the expression of the internal control gene (rpoD), using the 2^−ΔΔCt method. The sequences of the primers are listed in Table S1.

On day 21 after corresponding treatments, the Vglut2-cre^+/– mice were deeply anesthetized with 2% pentobarbital sodium (0.5 mL/100 g, intraperitoneal injection) and the L4–L6 spinal cord was immediately transferred to an ice-chilled lysis buffer through laminectomy. Total RNA was extracted from the spinal cord using Trizol reagent and reverse transcribed according to the manufacturer’s instructions (Takara, Tokyo, Japan). The expression of target genes was analyzed using the ViiA7 Dx system (Applied Biosystems, Carlsbad, CA, United States), with the SYBR Green qPCR Master Mix reagent system (Takara). The forward and reverse primers used in this study were: