Hiscript qrt supermix for quantitative pcr

The reverse transcription–polymerase chain reaction (RT–PCR) technology was employed to assess the mRNA expression levels of transforming growth factor TGF-β, IL-10, TNF-α, and IL-1β. Specimens from the left midbrain region were treated with the TRIzol kit (Takara Bio, Dalian, China) reagents to extract RNA, which was used to synthesize cDNA using HiScript QRT SuperMix for quantitative PCR (qPCR) (+gDNA wiper; R123-01; Vazyme Biotech Co., Ltd., Nanjing, China). The ChamQ universal SYBR qPCR master mix (Q711; Vazyme Biotech Co., Ltd.) was employed to run the RT–PCR reactions. The relative expression level of target mRNA was calculated using the 2^−ΔΔCT method, with Gapdh serving as the reference gene. Primers were generated by the Shanghai Biotechnology Service Co. (Shanghai, China) and had the following sequences:

 
Il1b forward 5′-AATCTCACAGCAGCATCTCGACAAG-3′ and reverse 5′-TCCACGGGCAAGACATAGGTAGC-3;

 
Tnfa forward, 5′-GCATGATCCGAGATGTGGAACTGG-3′ and reverse 5′-CGCCACGAGCAGGAATGAGAAG-3′;

 
Il10 forward, 5′-CAAGGCAGTGGAGCAGGTGA-3′ and reverse, 5′-CCGGGTGGTTCAATTTTTCATT-3′;

 
Tgfb forward 5′-GACCGCAACAACGCAATCTATGAC-3′ and reverse 5′-CTGGCACTGCTTCCCGAATGTC-3;

 
Gapdh forward: 5′-GACATGCCGCCTGGAGAAAC-3′ and reverse 5′-AGCCCAGGATGCCCTTTAGT-3′.

Extracellular matrix (ECM) media were purchased from ScienCell. RPMI-1640 media was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Trizol was purchased from Sigma-Aldrich (Sigma-Aldrich, Burlington, MA, USA). Hiscript QRT Supermix for quantitative PCR (qPCR) and AceQ qPCR SYBR Green was purchased from Vazyme (Nanjing, China). Antibodies (TRIM22, Beclin1, P62, ATG7, ATG5, mTOR, P-mTOR, AMPK, P-AMPK, ERK, P-ERK, and GAPDH) were purchased from Abcam (Abcam, Cambridge, UK). Age I and EcoRI were purchased from NEB (New England Biolabs, Ipswich, MA, USA). The BCA Protein Assay Kit was purchased from HyClone-Pierce, Inc (Thermo Fisher Scientific). Real-time fluorescence quantitative polymerase chain reaction (PCR) was purchased from ABI (StepOne PLUS, Thermo Fisher Scientific). The gel imager was purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). The ultraviolet–visible spectrophotometer was purchased from Thermo Fisher Scientific. The chemiluminescence imaging system used was a GE AI600. The microplate reader was purchased from Bio-Rad.

For RNA extraction, the strain R. anatipestifer ATCC 11845 was grown in TSB medium and TSB medium containing 40 μM EDDHA, 1 mM MnCl₂, 400 μM ZnSO₄, and 200 μM CuCl₂. R. anatipestifer ATCC 11845 and R. anatipestifer ATCC 11845 Δfur were grown in TSB medium. All the strains were grown at 37°C and 180 rpm until the exponential phase. Then, 6 × 10⁹ CFU (1 OD₆₀₀ = 2 × 10⁹ CFU) (22 (link)) of the cells was mixed with 1 mL RNAprotect bacterial reagent (Qiagen, 76506), and the total RNA was isolated using the RNeasy Protect Bacteria minikit (Qiagen, 74524) as described in a previous study (28 (link)). cDNA was synthesized from 1,000 ng template RNA with HiScript QRT SuperMix for quantitative PCR (qPCR) (Vazyme, R123-01). The cDNA was quantified by real-time PCR using SYBR green master mix (Vazyme, Q111-01) on a CFX Connect real-time system (Bio-Rad), and the mRNA levels of the feoA and feoB genes were normalized to those of 16S rRNA as described in a previous study (60 (link)). Experiments were performed in triplicate using three biological replicates.