Chamq universal sybr qpcr master mix

The expression levels of genes were quantified by qRT-PCR using gene-specific primers (Table S1) with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) on a CFX Connect^TM Real-Time System (Bio-Rad, Hercules, California, America). A PCR reaction mix contained 10 μL of ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China), 125 nM of each forward and reverse primer, 2.0 μL of cDNA template, and 7.5 μL of RNase-free water. Data analysis was calculated by the 2^−ΔCt method or 2^−ΔCtΔCt method [36 (link)]. All experiments were measured in three biological replicates.

Total RNA from the cecal tissues (about 50 to 100 mg) was extracted by the addition of 1 mL of MagZol-reagent (#R4801-02; Magen Biotechnology, Guangzhou, Guangdong, China) according to the manufacturer’s instructions. The concentration and purity of the total RNA were assessed using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). Subsequently, the RNA was reverse transcribed to cDNA using the ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO, OSAKA, Japan) according to the manufacturer’s instructions. The cDNA samples were amplified by real-time quantitative polymerase chain reaction with ChamQ Universal SYBR qPCR Master Mix from Vazyme Biotechnology (Nanjing, Jiangsu, P. R. China). Gene-specific primers for each gene were designed using Primer3web, version 4.1.0 (Supplementary Table 1). PCR was performed on the C1000 Touch PCR Thermal cycler (BIO-RAD Laboratories, Shanghai, China) using ChamQ Universal SYBR qPCR Master Mix from Vazyme Biotechnology (Nanjing, Jiangsu, P. R. China) and was as follows: 40 cycles of 95°C for 15 s and 60°C for 30 s. All measurements will be performed in triplicate. The messenger ribonucleic acid (mRNA) expression of target genes relative to beta-actin (β-actin) was calculated using 2^-ΔΔCT method (41 (link)).

Total RNA was isolated from tissues and sperm using the TRIzol Reagent according to the manufacturer’s instructions. RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) and a Q7 real-time PCR System (Applied Biosystems) according to the manufacturer’s instructions. Gapdh was employed as an endogenous control, and the threshold cycle (Ct) for each test was established.
For sRNA analyses, 100 ng of total RNA was reverse transcribed to cDNA using a PrimeScript™ RT Reagent Kit (Takara, Japan) and stem-loop RT primer (Tsingke Biotechnology Co., Ltd.). RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) and a Q7 real-time PCR System (Applied Biosystems). The relative expression of sRNA was normalized to U6 snRNA. The employed primers are listed in Supplementary Tables S5 and S6.

To verify transcriptome sequencing data, three DEGs in each of the hippocampal brain regions of short- and long-term SM were selected for validation by quantitative real-time PCR (RT-qPCR), as previously mentioned (Li et al., 2020 (link); Ding et al., 2021 (link)). Specific primers were designed according to reference sequences in the NCBI database with Primer Bank and are listed in Table 1. The primers were synthesized by Sangon Biotech (China). Total RNA was extracted from the HPC using TRIzol reagent and reverse-transcribed into cDNA using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (#K1622, Thermo, USA). After reverse transcription, RT-qPCR was carried out on Applied Biosystem Real-Time PCR System (CFX96, Bio-Rad, USA). The 15 μl reaction system contained 7.5 μl of 2x ChamQ Universal SYBR qPCR Master Mix (#Q711, Vazyme, China), 0.3 μl of each primer (forward and reverse), 5 μl of cDNA template, and 1.9 μl of nuclease-free deionized water. The three-step method was performed for amplification with denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and 60°C for 30 s; and final elongation at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The relative mRNA expression levels of selected genes were calculated with the 2^–ΔΔCq method using GAPDH as an internal reference. Each sample was tested in triplicate.