Pcr master mix

Total RNA was extracted from the liver with Trizol reagent (Life Tech, USA). First-strand cDNA was generated from 3 μg of total RNA in a volume of 20 μL using a reverse-transcription kit (TIANGEN BIOTECH, BJ CHN) with Oligo dT as a primer. The PCR was performed in a final volume of 25 μL containing 2 μg of cDNA, 9.5 μL of double-distilled water, 12.5 μL of PCR master mix (TIANGEN BIOTECH, BJ CHN), and 0.5 μL of each of the primers specific for PPAR-α or GADPH. The primer sequences are as follows: (1) PPAR-α sense primer, 5’-ATGTCCGTGGAGACCGTCA-3’; antisense primer, 5’-GGTTCTTAAGGAACTCGCGTG-3’; (2) GADPH sense primer, 5’-AGGCCGGTGCTGAGTATGTC-3’; and antisense primer, 5’-TGCCTGTTCACCACCTTCT -3’. PPAR-α was amplified under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s in a GeneAmp PCR system 9700 (PE, USA). As an internal control, GADPH was amplified concomitantly using the same amplification conditions. PCR products (5 μL) were electrophoresed on a 2.0% agarose gel. Band intensity was quantified under UV light using the Sygene Bio-ID system (USA). The level of mRNA expression was expressed as the ratio of band intensity of the target gene relative to that of GADPH.

cDNA extracted from ticks was examined using polymerase chain reaction (PCR) targeting the rrs gene, and the amplified gene fragments were sequenced to detect and identify Rickettsiales bacteria in the ticks. For further identification of CRT, the rrs-positive tick samples were further amplified by PCR targeting the gltA, groEL, ompB and Sca4 genes and sequenced. DNA samples were considered CRT-positive when at least two of these four genes were positive.
DNA was amplified using a system of 20 μl, including 11 μL PCR Master Mix (TianGen, China), 1 μl of DNA from each sample, and 0.5 μl each of reverse and forward primer, and filled to volume with double-distilled water. Negative and positive controls were included in each PCR. The amplified PCR products were separated on a 1.5% agarose gel and purified using the Gel DNA Recovery Kit (TianGen, China) according to the manufacturer’s instructions for sequencing. Oligo7 (https://www.oligo.net/downloads.html) was used to design specific primers. The synthesis of primers and sequencing were performed by Sangon Biotech (Shanghai, China). The primers used for amplification are listed in Table 2.

Human VSMCs were supplied by Modern Analysis and Testing Center of Central South University. Human embryonic lung fibroblasts (HLF), human HCMV strain AD169, DH-5α, pEGFP-C1 were preserved by Department of Microbiology, Central South University. DMEM and DMEM/F12 (1:1) medium were purchased from Hyclone, USA. Fetal bovine serum (FBS) was from GIBCO. LB broth was from Tianhe Co., Ltd, Hangzhou, China. Ampicillin and kanamycin were from Amresco, USA. Plasmid extraction kit and DNA gel extraction kit were supplied by OMEGA, USA. pGM-T vector kit and PCR master mix were from Tiangen (Beijing) Co., Ltd. TRIzol and Lipofectamine LTX and PLUS reagents were from Life Technologies, USA. cDNA first strand synthesis kit was from Toyobo (Shanghai) biological Technology Co. CCK 8 cell proliferation test kit was supplied by Dongren chemical technology (Shanghai) Co., LTD. Hoechst33342/PI double dye cell apoptosis detection kit was from Beibo (Shanghai) biological technology Co., LTD.

The PCR was performed according to the methods of Weir et al. (2012) (link). All the PCR reactions were conducted in 25-μl volumes containing 12.5 μl PCR MasterMix (TIANGEN BIOTECH (BEIJING) CO., LTD., Beijing, China), 10 μM primers (both), 1 μl of the template DNA (20 ng μl^-1), and 9.5 μl of double-distilled H₂O. The PCR program to amplify ACT and GAPDH included a denaturation step at 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 59°C for 30 s, and 72°C for 2 min. The final cycle comprised 72°C for 10 min. The amplifications of ITS, TUB2, CHS-1, and CAL were also performed similarly with annealing temperatures of 56, 58, 58, and 57°C, respectively. The amplification products were analyzed on a 1.0% agarose gel in tris/borate/EDTA buffer. The PCR products were then purified and sequenced at Hangzhou Shangya Biotechnology Co., Ltd (Zhejiang, China).

K. pneumoniae virulence genes were detected by PCR. The extracted genomic DNA from planktonic isolates served as a template for the amplification of virulence genes and for determining capsular serotypes. All PCR primer sequences and corresponding references are listed in Table S2. PCR amplification was performed in a total volume of 50 μl, containing 2 × PCR Master Mix (Tiangen Biotech Beijing Co., Ltd, Beijing, China), 0.5 mM of each primer, and 1 μl template DNA. The cycling conditions were as follows: 95°C for 3 min, followed by 30 cycles at 95°C for 30 s, 49°C−58°C for 30 s, 72°C for 60 s, and a final 10-min extension step at 72°C. Each PCR set included a no-template control and a positive control [NTUH-K2044 strain for magA(K1) and wcaG]. Amplification products were analyzed by electrophoresis in 1.0% agarose gels.