High capacity reverse transcriptase kit

Lung samples were stored at -80°C in an RNA stabilization solution. RNA was extracted with a commercial kit (mirVana Kit, Ambion, USA) and cDNA was transcribed using a High-Capacity Reverse Transcriptase Kit (Applied Biosystems, USA). Real-time polymerase chain reaction (PCR) was performed with SYBR^®Green PCR Master Mix (Applied Biosystems, USA) and SYBR^®Green primers (Applied Biosystems, USA) against β-actin, C3, C3aR, decay-accelerating factor (DAF), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10, IL-6, and Caspase-3 (Table 1). Targets were amplified over one cycle for 2 min at 50°C, one cycle for 10 min at 95°C, 40 cycles for 15s at 95°C, and for 1 min at 60°C. Relative gene expression was determined in relation to reference values obtained from sham animals (n=5).

For qRT-PCR validation of select differentially expressed genes, RNA samples were collected in the same manner as for RNA sequencing, from E. amylovora cultures grown in LB medium and then induced for 18 h in HIMM. Three replicate RNA isolations were made from independent bacterial cultures. 500 ng of total RNA was used as template for reverse transcriptase reactions using the High-Capacity Reverse Transcriptase kit (Applied Biosystems, Foster City, CA, United States) following prescribed protocols. Resulting cDNA was utilized as template in qRT-PCR reactions set up using SYBR green 2X master mix (Applied Biosystems, Foster City, CA, United States) according to manufacturer’s protocols and run on an Applied Biosystems StepOnePlus instrument. The housekeeping gene recA was included as an endogenous control, and relative mRNA abundance was calculated using the 2^–ddCt method (Livak and Schmittgen, 2001 (link)).

RNA isolation, cDNA synthesis, and real-time PCR were performed as previously described (Sougiannis et al., 2019 (link)) using reagents from Applied Biosystems (Foster City, CA, United States). Briefly, RNA was extracted from the gastrocnemius using the TRIzol/isopropanol/chloroform procedure (Life Technologies, GIBCO-BRL, Carlsbad, CA, United States). RNA sample quality and quantities were verified using a Nanodrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and determined to be of good quality based on A260/A280 values (>1.8) prior to cDNA synthesis using High capacity Reverse Transcriptase kit (Applied Biosystems, Foster City, CA, United States). Probes for MCP-1, IL-6, IL-1β, IL-10, TNF-α, IFNγ, CD11c, CD206, F4/80, and CD68 as well as housekeeping genes Hmbs, B2M, TBP, H2afv, and 18s were purchased from Applied Biosystems (Foster City, CA, United States). Quantitative RT-PCR analysis was carried out as per the manufacturer’s instructions (Applied Biosystems, Foster City, CA, United States) using Taq-Man Gene Expression Assays on a Qiagen Rotor-Gene Q. Data were normalized to vehicle treated controls and compared to five reference targets (Hmbs, B2M, TBP, H2afv, and 18s), which were evaluated for expression stability using GeNorm (St-Pierre et al., 2017 ).

Total RNA was extracted from 10 mg skin tissue after lysis and homogenization, using silicate gel technique provided by the RNeasy Mini Kit (Qiagen) [65 (link)]. The concentration and purity of RNA were measured at 260, 280 and 230 nm using Nano Drop 2000c spectrophotometer (Thermo Scientific, USA). A ratio of A₂₆₀/A₂₈₀ = 1.8–2.1 and A₂₆₀/A₂₃₀ = 1.8–2.1 indicates highly pure RNA. Total RNA was reverse transcribed into cDNA using high-capacity reverse transcriptase kit (Applied Biosystems™, USA, catalog no. 4368814). To detect TP53 in tissue samples, primers had been matched to the mRNA sequences of the target genes (NCBI Blast software). GADPH was used as housekeeping gene [66 (link)].

Approximately 50–100 mg of tissue was homogenized in 1 ml TRIzol (TakaRa Biotechnology, Dalian, China) and the total RNA was extracted according to manufacturer’s instructions. The quality and quantity of RNA were determined by the Nanodrop (Thermo Scientific, ND-2000, USA), with 260/280 ratio >1.8. Total RNA was reverse transcribed with a High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA). The primers were designed with Primer3 software and listed in
Table 1．
The 15 µL PCR reaction mix contained 3 µL of cDNA (10 ng/µL), 7.5 µL of iQ
^TM SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), 0.5 µL of primer mix (10 µM each), and 4 µL of ddH
₂O. After 5 min denature at 95°C, 40 cycles were performed: annealing and extension at 60°C for 45 seconds and denature at 95°C for 10 seconds. Dissociation curve was performed after finishing 40 cycles to verify the quality of primers and amplification. Relative expression of genes was calculated by the 2
^-ΔΔCt method and normalized to the house keeping gene β-actin or expressed as a percentage of controls
^{8 (link),
21 (link)}
.

For SAMHD1 mRNA quantification, total cellular RNA was extracted using the RNeasy mini kit (Qiagen, Limburg, the Netherlands) according to the manufacturer’s guidelines. One hundred nanograms of the total RNA from each cell type was used as a template for first-strand cDNA synthesis using high-capacity reverse transcriptase kit (Applied Biosystems, Waltham, MA, USA, #4368814). SAMHD1 mRNA was quantitated by SYBER Green-based quantitative real-time PCR (Stratagene-3000; Stratagene, La Jolla, CA, USA) analysis was performed using the specific primers. Quantification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was carried out for normalization. Relative expression of mRNA species was calculated using the comparative C_T method.