Linegene 9600 plus machine

Bacteria were grown in 1/10 TSB medium at 28°C for 24 h in 60 mm dishes (Corning). The supernatant was discarded and the bacteria accumulated in the biofilms under the dishes were scraped. The total RNA was extracted using a Bacterial RNA Kit (Omega). The cDNA was synthesized using a PrimeScript RT reagent Kit with gDNA Eraser (Takara) and quantified via TB Green Premix Ex Taq (Takara). The gene transcript levels were tested in triplicate for real-time PCR in a Linegene 9600 Plus machine (Bioer). Primer pairs of Qgyrb-F/R, QcsgD-F/R, QcsgA-F/R, QadrA-F/R, and QbcsA-F/R (Table 3) were used for the mRNA detection of gyrB, csgD, csgA, adrA, and bcsA, respectively. The target genes' mRNA levels were normalized to the gyrB mRNA levels (2^−ΔΔCt) (30 (link)).

RNA extraction, complementary DNA (cDNA) synthesis, and RT-qPCR were conducted in accordance with our previous studies [76 (link)]. Briefly, cDNA was made using the SuperiorScript III cDNA synthesis kit (#EZ405S, Enzynomics, Daejeon, Republic of Korea). The cDNA was diluted with RNase-free water to a final concentration of 8 ng/µL, and the samples were kept at −80 °C. RT-qPCR was carried out using TOPreal^TM SYBR Green qPCR PreMix (#RT500M, Enzynomics, Daejeon, Republic of Korea) and the LineGene 9600 Plus machine (BIOER, Hangzhou, China) following the manufacturer’s instructions. The primers for RT-qPCR are shown in Table 1. The annealing temperature for the reaction was 58 °C, and the built-in software created the amplification curves and calculated the threshold cycle values. The GAPDH reference gene was used to normalize all the readouts. Data were reported as the mean relative values compared to the CON group using the 2^−∆∆CT method.

The hippocampi (n = 8 mice/group) were collected and digested in Trizol reagent for RNA extraction based on the manufacturer’s instructions (RNAeasy Lipid Tissue Mini Kit; Qiagen). Reverse transcription was performed using the Superscript™ II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). The resulting complementary DNA (cDNA) was diluted with RNase-free water to achieve a final concentration of 8 ng/μL, and the samples were stored at −70 °C. The quantitative PCR was performed with TOPreal™ qPCR 2XPreMIX (TaqMan Probe) (Enzynomics, Daejon, Korea) using the LineGene 9600 Plus machine (BIOER, Hangzhou, China) according to the manufacturer’s instructions. The qRT-PCR primers are listed in Supplementary Table S3. The annealing temperature for the reaction was 58.5 °C, and the built-in software generated the amplification curves and computed the threshold cycle values. All the readouts were normalized using the β-actin reference gene. Via the 2^−ΔΔCT method [64 (link)], data were expressed as mean relative values compared to the values of the WT controls.