Thermofisher quantstudio 3 rt pcr well q3

The expression levels of the genes identified in the Dap-seq experiment were further analyzed using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from M5-90 or M5-90 irr mutant cells with TRIzol (CWBIO, Beijing, China). Each group has three replicates. Briefly, the RNA concentration and quality were evaluated using a Nanodrop 2000 spectrophotometer (Thermo Fisher, USA). The extracted RNA was reverse-transcribed to cDNA using a First Strand cDNA Synthesis Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. qRT-PCR was performed using SYBR (CWBIO, Beijing, China) and a ThermoFisher QuantStudio 3 RT PCR-Well Q3 (Thermo Fisher, USA). The primers for the target genes are listed in S1 Table. The reaction conditions used are as follows: initial denaturation at 95°C for 5 min followed by 46 cycles of 95°C for 30 s and annealing at 56°C or 60°C for 30 s. The data were normalized according to the expression level of the 16S ribosomal RNA and the expression level of each gene was calculated using the 2^-ΔΔCT method.

Total RNA was isolated from RAW264.7 cells (infected with B. melitensis Y3 or B. melitensis M5-90) at different points in time using an RNeasy Kit (CWBIO, Beijing, China). DNA was eliminated using a TURBO DNA-free DNAse (Ambio). The concentration and quality were assessed via a Nanodrop 2000 (Thermo, USA). RNA samples were subjected to reverse transcription using a HiFiScript cDNA Kit (CWBIO, Beijing, China) according to the manufacturer’s instructions. Real-time PCR analysis was performed using ThermoFisher QuantStudio 3 RT PCR-well Q3 (Thermo Fisher, USA) with SYBR (CWBIO, Beijing, China). The primers for target genes were as follows: 16 S forward, 5′-ACTAAGGGCGAGGGTTGC-3′; 16 S reverse, 5′-CACTGGACCATTACTGACGC-3′; BtpA forward, 5′-GCCCGCAAGAGAATTAGATGGACTG-3′; BtpA reverse, 5′-GAGGGACTGAAACGCCGAACTTC-3′; vjbR forward, 5′-CCGCTACGTAACGCATACCTATCG-3′; vjbR reverse, 5′-CAGGTAGCAGGCAGCGTCATAAG-3′. The reaction conditions were as follows: initial denaturation at 95°C for 5 min followed by 45 cycles of 95°C for 30 s and annealing at 57°C or 60°C for 30 s. Fold changes of each gene were calculated using the 2^-ΔΔCT method and the mRNA levels were normalized according to 16 S ribosomal RNA expression.