Sybr green master mix

The qRT-PCR template cDNA was synthesized from total RNA. Each qRT-PCR reaction system had a total volume of 20 μL, including 2 μL of c DNA, 4 μL of upstream and downstream primers, 7.2 μL of ddH₂O, and 10 μL mixture of 2 × SYBR Green Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China). All qRT-PCR reactions were carried out in a CFX96 Real-Time System (Bio-rad Laboratories, Inc., Berkeley, CA, USA), referring to the reaction parameters in the SYBR Green Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) guide. The actin gene was used as internal reference gene, and the relative gene expression level was calculated by the 2^−△△CT method [31 (link)].

Total RNA was isolated from the GA muscle using RNAsio Plus (9109, TAKARA, Kyoto, Japan) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from 2000 ng total RNA with the HiScript II Q Select RT SuperMix (R223-01, Vazyme, Nanjing, China). qRT-PCR was performed using a reaction mixture containing SYBRTM Green master mix (Q711-02-AA, Vazyme, Nanjing, China). Results were calculated using the 2-△△CT relative quantification method normalized to the 18S gene. The sequences of the primer pairs are shown in Supplementary Table S1.

Total RNA was extracted using TRIzol® Reagent (Thermo Scientific, Waltham, MA, United States) and reverse-transcribed with oligo-DT using HiScript^TM Reverse Transcriptase (Vazyme, Beijing, China) according to the manufacturer’s instructions. The primers for PTGS1, PTGS2, DRD2, stem cell factor (SCF) and c-kit were synthesized by Tsingke (Beijing, China). GAPDH was used as an internal reference gene. The 2^−ΔΔCT method was used to determine the relative expression of each gene. The primers used in this study are provided in Supplementary Table S1.
qRT‒PCR was performed using SYBR^TM Green Master Mix (Vazyme) with a QuantStudio 6 Flex system (Applied Biosystems, Foster City, CA, United States). The PCR cycling profile was as follows: one cycle of 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Fluorescence signals were detected using the QuantStudio 6 Flex system. Gene expression was normalized to that of the endogenous control GAPDH. The 2^−ΔΔCT method was used to determine the relative expression of each gene.

Briefly, total RNA was extracted from tissues of treated and untreated animals by the trizol isolation reagent according to the manufacturer's instructions. After some precipitation, separation and purification, the RNAs were reverse- transcribed into cDNA with revert aid first strand cDNA synthesis kit (K1622, Fermentas, Lithuania). The primers for PCR were listed in Table S1. According to the manufacturer's guidelines, real-time PCR was performed with the 2×SYBR green mastermix (Q111-01, Vazyme, China). The results were calculated via the 2^-ΔΔCt method using the threshold cycle (Ct) value, and reported as fold change in gene expression.

To validate the RNA sequencing (RNA-Seq) results, four genes were randomly selected for a quantitative reverse transcription PCR (qRT-PCR) analysis using a 2× SYBR GREEN Master Mix (Vazyme Biotech Co., Ltd.). Primers were designed using the NCBI database, and β-actin was used as the reference gene. The thermal cycle of SYBR Green RT-PCR was as follows: 95°C for 10 min and 40 cycles at 95°C for 10 s and 60°C for 30 s. The genes to be validated are listed in Table 1, whereas the primers corresponding to the genes to be verified are listed in attachment.

RNA was extracted and reverse-transcribed to cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme, China) or All-in-One^TM miRNA First-Strand cDNA Synthesis Kit (Genecopoeia, United States). sigA gene or MysA was used as an internal reference. The primers for amplification of selected genes are listed in Supplementary Table 1. RT-PCR was performed in 10 μl reaction mixtures, including 5 μl of 2 × SYBR Green Master Mix (Vazyme), 10 μM of forward and reverse primers, 0.2 μl Dye 2, 1.5 μl cDNA template (1:10 diluted), and double-distilled water to a final concentration of 10 μl. Samples and standards were run in triplicate in an ABI 7500 thermocycler (Applied Biosystems, United States) and analyzed using model 7500 SDS software version 1.3.1. PCR was carried out at 94°C for 10 min and then 94°C for 15 s and 60°C for 1 min for a total of 40 cycles.