Quantstudio 6 flex system

Total RNA was extracted from SK-Hep1 cells using an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA concentrations were determined via a NanoDrop® (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). Quantitative PCR was performed using SYBR Green Master Mix (Thermo Fisher Scientific) in a total volume of 20 µL with the QuantStudio 6 Flex system (Thermo Fisher Scientific). The expression of the target genes was normalized to β-actin expression, and relative quantification was evaluated with the 2 − ΔΔCt method.

Total RNA was extracted from differentiated C2C12 myotubes or gastrocnemius muscle tissue using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc). Quantitative PCR was performed using SYBR Green Master Mix (Thermo Fisher Scientific) in a total volume of 20 µL with the QuantStudio 6 Flex system (Thermo Fisher Scientific). The expression of the target genes was normalized to GAPDH expression, and relative quantification was evaluated with the 2^−ΔΔCt method. The primer pairs for the specific target genes were designed as listed in Table S1.

Cells were lysed by TRIzol Reagent (Invitrogen) to obtain RNA according to the manufacturer’s protocol. Reverse transcription was performed with 1 µg RNA using HiScript II Q RT SuperMix (Vazyme Biotech Co., Ltd). qPCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) on QuantStudio 6 Flex system (Thermo Fisher Scientific). Data were analyzed using a 2–ΔΔCt method.

Bacteria were grown in LB medium with or without 50 mg/L of kanamycin and 250 μM IPTG to an OD₆₀₀ of 0.5. The culture (0.5 mL) was removed and mixed with 1 mL RNAprotect bacterial reagent (Qiagen, Hilden, Germany), and RNA was isolated using a RNeasy minikit (Qiagen). After RNase-free DNase (Invitrogen) treatment, cDNA was synthesized from 1 μg of template RNA using Omniscript Reverse Transcription reagents (Qiagen) and random primers (Invitrogen). The amount of cDNA was quantified by qRT-PCR using the TB Green Premix Ex Taq (TaKaRa) with a QuantStudio 6 Flex System (ThermoFisher Scientific). The ΔΔC_T values of the target genes were calculated using the C_T values of the reference strains and the housekeeping gene rpoB. To detect the cDNA corresponding to dam, recC, umuD, phoU, glpD, sucB, recA, and fis, and rpoB mRNAs, the primer pairs presented in Table S2 were used, respectively.

Total RNA from peripheral blood was extracted using the UNlQ-10 Column Trizol Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to check the concentration and purity of the extracted RNA, with the A260/A280 between 1.8 and 2.0. The cDNA synthesis was conducted using Hifair Ⅲ 1st Strand cDNA Synthesis SuperMix (Yeasen Biotech, Shanghai, China). Using β-actin as a reference, we performed quantitative RT-PCR with Hieff qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China) in the QuantStudio 6 Flex system (Thermo Fisher Scientific, Waltham, MA, USA). Primer sequences (Sangon Biotech, Shanghai, China) for reference and candidate genes are shown in Table 1. The 2^−ΔΔCt method was applied to calculate the relative expression level of mRNA.

In the study, total RNA from peripheral blood was extracted using a UNlQ-10 column TrizolTotal RNA isolation kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions.
We checked the concentration and purity of the extracted RNA using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Among them, A260/A280 was controlled between 1.8 and 2.0. This study applied Hifair III1st Strand cDNA synthesis SuperMix (Yepsen Biotech, Shanghai, China) for cDNA synthesis. In this study, we used GAPDH as a reference. We performed quantitative RT-PCR by using Hieff qPCR SYBR Green MasterMix (Yepsen Biotech, Shanghai, China) in a Quant Studio 6 Flex system (Thermo Fisher Scientific). Primer sequences (Sangon Biotech) used for reference and candidate genes were shown in Table 1. We applied the 2^−∆∆Ct method to calculate the relative expression levels of mRNAs.

Total RNA was isolated as described above and cDNA was synthesized using the SuperScript III Reverse Transcriptase with Oligo dT primers (Thermo Fisher Scientific). qRT-PCR analysis were performed as previously described^{47 (link)} on a QuantStudio 6 Flex system (Thermo Fisher Scientific) using SYBR Green Master Mix (Thermo Fisher Scientific). Primers used are listed in Supplementary Dataset 3. Gene expression values were calculated using Ct values and normalized using the following housekeeping genes: P. polyphylla, closest homolog to Hordeum vulgare alpha tubulin U40042.1^{48 (link)}; T. foenum–graecum, closest homolog to Medicago truncatula Medtr3g091400.1^{49 (link)}.