Hiscript 2 q rt supermix with gdna wiper

Total RNA from knockout cell lines was isolated using the TRIzol reagent (Invitrogen, 15596018) and 1 μg of total RNA, in 20 μL mixed reverse‐transcription reagent, was reverse transcribed using HiScript II Q RT SuperMix with gDNA wiper (Vazyme, R223‐01). The specific primer pairs (Table S3) were used for qPCR amplification utilizing the ViiA™ 7 Real‐Time PCR System (Applied Biosystems). The qPCR reaction includes 1 μL of cDNA templates, 10 μmol/L of each specific primer and 10 μL 2 × ChamQ SYBR qPCR Master Mix (Vazyme, Q331‐02). The reaction procedure was set as follows: 95°C for 3 minutes and 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. Relative quantification of the target gene expression was calculated using 2^−ΔΔct method. Each reaction was performed in triplicate, and the data were calculated as M ± SD.

Gene expression analysis was done using species-specific QRT-PCR. Primers were designed using NCBI’s Primer-BLAST [22 (link)]. Primer sequences are shown in Table S3. RNA was extracted from tissues and cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using HiScript II Q RT SuperMix with gDNA wiper (Vazyme, China) following the manufacturer’s instructions. QRT-PCR was performed on a CFX96 Touch Real-Time PCR system (Bio-Rad, USA), using ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The QRT-PCR protocol was as follows: 1 cycle of 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Samples were analyzed in triplicate, and the relative gene expression was determined using the 2^−ΔΔCt method using beta-actin as a reference gene [23 (link)]. Statistical significance between two groups was determined via Student’s t-test on GraphPad Prism (v6.01). p < 0.05 indicated statistical significance.

Total RNA was isolated using RNeasy Plus Mini Kit (74134, Qiagen), cDNA was generated using HiScript II Q RT Super Mix with gDNA wiper (R223, Vazyme Biotech), and qPCR was performed using the AceQ qPCR SYBR Green Master Mix (Q111, Vazyme Biotech). Relative quantification was performed using the comparative Ct method with normalization to Actin. Primer sequences are available in Supplementary Table S2.

Bacterial cells were cultured in 1/10 TSB and collected at an OD₆₀₀ of ~1.0. RNA extraction was carried out with a bacterial RNA extraction kit (Yeasen MolPure, China). The concentration and quality of RNA were detected with an Eppendorf BioPhotometer Plus. cDNA synthesis was performed with 250 ng of RNA using a kit (Vazyme HiScript II Q RT SuperMix with gDNA wiper). Primers for qRT-PCR designed with primer 3 online are shown in Table S1, and the 16S rRNA gene was used as a reference. The qRT-PCRs was performed on a Quantstudio 6 Flex system (Applied Biosystems) using ChamQ SYBR qPCR master mix (Vazyme). The experiment was repeated three times, each time in triplicate. Relative expression was analyzed using the threshold cycle (2^−ΔΔCT) method.