Iq5 real time pcr detection system

Total RNA was extracted from cells or tissues using a trizol reagent (Invitrogen, Carlsbad, CA, USA). The total RNA (1 μg) of every sample was reverse transcribed using the Fast-King RT kit (Tiangen Biotech, Beijing, China). The Bio-Rad iQ5 real-time PCR detection system was used with a Talent qPCR PreMix SYBR Green kit (Tiangen Biotech) according to the manufacturer's instructions. Relative expression of each target gene was determined by normalization to the expression of GAPDH. The primers were synthesized by the Shanghai Sangon company, and the primer sequences were shown in Table 1.

Total cellular RNA was extracted from cells grown for 3 h, 9 h, 21 h and 24 h using Trizol reagent (Invitrogen). Reverse transcription step was carried out using Goldenstar RT6 cDNA Synthesis Kit Ver 2 (TsingKe, Chengdu, China) with random primer mix following the manufacturer’s manual. Quantitative real-time reverse transcription PCR was performed in the Bio-Rad iQ5 real-time PCR detection system with SuperReal PreMix (SYBR Green) (TIANGEN, Beijing, China). All optimized primers are shown in Table S4 and were designed using primer software to amplify approximately 100 bp from the 3′ end of the target genes. PCR conditions were 1 min at 95 °C, followed by 40 cycles of heating at 95 °C for 15 s and 60 °C for 15 s, and 72 °C for 30 s, and final extension at 72 °C for 5 min. PCR amplification was detected by SYBR Green. The ratios of the cycle threshold (Ct) values were determined from the Bio-Rad iQ5 Optical System Software provided. To analyze the gene expression level, the ΔΔCt method was chosen and standard curves of each primer were plotted to ensure similar amplification efficiency compared with the reference gene. The rrsA gene, encoding the 16S RNA, and tdh2 gene served as an endogenous control to normalize for differences in total RNA for Z. mobilis 8b, and S. stipitis, respectively.