Rt reagent kit

Total RNA was extracted from the samples using Trizol reagent (Thermo Fisher Scientific) following the manufacturer's instructions. Subsequently, cDNA was synthesized using a reverse transcription (RT) reagent kit (Vazyme, China). Quantitative PCR was performed on the PowerUp SYBR Green Master Mix kit (Vazyme). The primer sequences used are provided in Table S2, and GAPDH was used as internal reference for mRNA quantification. The relative expression levels of the target mRNAs were calculated by the 2^−ΔΔCt method.

Total RNA was obtained from cultured cells with TRIzol reagent (Invitrogen, USA) as instructed by the manufacturer. Quantitative real-time RT-PCR detecting mature miR-30e was carried out in triplicate with RT Reagent Kit (Vazyme, Nanjing, China) as directed by the manufacturer, withAceQ SYBR Master Mix (Vazyme, Nanjing, China) on a 7900HT system. MicroRNA-30e levels in each group were determined relative to U6 amounts, by the 2^−ΔΔCt) method.

Trizol (Invitrogen, CA, USA) was used to isolate sample RNA according to provided direction. The stem-loop-specific primer approach was used for assessing miR-22 expression, with U6 used for normalization. Twist1 expression was assessed via using the RT Reagent Kit (Vazyme, China) to reverse transcribe total RNA, and GAPDH was used for normalizing gene expression. SYBR Green Master Mix (Vazyme, China) was used for all qRT-PCR reactions, with relative quantification (2^−ΔΔCt) used for all fold change calculations.

RNA of glioma cells or tissues grinded into debris was extracted with Trizol reagent following the product instruction (Invitrogen, Gaithersburg, MD, USA). RNase R was utilized to confirm the stability of circSMO742 and eliminate SMO mRNA. Stem-loop-specific primer method was applied to measure expression levels of miR-338-3p. U6 was adopted as control. QRT-PCR was performed via the SYBR Select Master Mix in an ABI Prism 7000 Sequence Detection. To examine the SMO mRNA levels, reverse transcription for total RNAs was carried out by oligodT primer using RT Reagent Kit (Vazyme, Nanjing, China). The relative expression levels were evaluated with relative quantification (2^−ΔΔCt). The primer sequences were designed according to the data deposition in public repository and shown in Table 2. SMO, NCBI, NM_005631.4; miR-338-3p, miRBase, MIMAT0000763; circSMO742, circBase, hsa_circ_0001742.

To extract the total RNA of the three different strains of bacteria (wild-type SCCF01, Δcrp, and C-crp), a Total RNA Extraction Kit (FOREGENE Biotech Chengdu Co., Ltd., Chengdu, China) was used. Subsequently, to determine the total RNA integrity and density, an RT reagent Kit (Vazyme Biotech Nanjing Co., Ltd.) was used for reverse transcription.
In a fluorescent quantitative PCR (q-PCR) system, 10 μL of ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Nanjing Co., Ltd., Nanjing, China), 1.6 μL of diluted cDNA, 0.4 μL of each primer, and 7.6 μL of nuclease-free water were used in a 20 μL reaction. The reaction protocol consisted of 2 min at 95 °C, 40 cycles of 10 s at 95 °C, and 30 s at 60 °C. Every sample was examined three times. The 2^−ΔΔCT technique was used to determine the relative expression of genes; the 16S gene was chosen as a standardized internal reference. The virulence factor primers related to the phenotype experiments were designed according to the complete genome data of V. mimicus SCCF01 available in our laboratory. Primers are listed in Table S2.