Steponeplus real time pcr system

HEK-293T cells (4 × 10⁵ cells) were seeded in 6-well plates and transfected with a reporter plasmid (pMIR-RNL-TK) overexpressing SPAG7. After 24 h, cells were transfected with 20 µl of PolyFect™ Transfection Reagent (Qiagen) and incubated at 37 °C and 5% CO2 for 48 h. Following incubation, RNA was isolated using the miRNeasy^® Mini Kit, cDNA was generated using TaqMan^® MicroRNA Reverse Transcription Kit, and RT-qPCR was performed using TaqMan^® Small RNA Assay for each miRNA assay. All RT-qPCR reactions were set up using a QIAgility automated PCR setup robot (Qiagen) and detected using StepOnePlus™ Real-Time PCR System.

The inflammatory array used in this study, RT² Profiler PCR Array, targeted 84 human inflammatory cytokines and receptors (Qiagen, PAHS-011ZC, 330231). HMC3 cells were seed in 12-well plates at a density of 1E5 cells/mL. Cells were pre-treated with either peptide (10 gμ/mL) or 1% water for 2 h. Subsequently, cells were infected with TC-83 (MOI = 2) and incubated for 1 h at 37 °C. The viral inoculum was removed from cells and cells were re-treated with respective peptide or 1% water and incubated at 37 °C. Cells treated with lipopolysaccharide (LPS) were used as a positive control. At 16hpi, cells were lysed with TRIzol and RNA was extracted using Zymo Direct-Zol Miniprep kit. cDNA was synthesized using RT² First Strand Kit (Qiagen, 330401). The PCR array was quantified as per manufacturer’s instructions using RT² SYBR Green qPCR master mix (Qiagen, 330521) and StepOnePlus Real-Time PCR system.

For RNA isolation, C. elegans at the same stage (L1-L2) grown on indicated food plates were quickly collected from NGM plates and washed three times with M9 buffer, followed by the addition of 250 ul TRIzol (Invitrogen). Lysates were preserved at 80°C until RNA isolation. RNA was isolated with two chloroform extractions (50 μl each), followed by isopropanol precipitation (125 μL) of the aqueous phase, and a single wash of the resulting pellet with 70% ethanol (250 μL). RNA pellets were dried in a tissue culture hood and re-suspended in RNase-free water, and then purified of contaminating DNA by DNaseI treatment (TURBO DNA-free Kit, Invitrogen) followed by cleanup using QIAGEN RNeasy columns.
cDNA for RT-PCR experiments was synthesized using 300 ng of total RNA template, SuperScript III, and oligo(dT)12–18 primer, according to the manufacturer’s protocol (Life Technologies). qRT-PCR reactions were performed in triplicate using the Applied Biosystems StepOnePlus Real-Time PCR system and Rotor-Gene SYBR Green PCR Kit (QIAGEN) and fold-change calculations were performed manually. A Student’s t test was used to evaluate statistical significance. Primers can be found in Supplementary file 1.

In the appropriate amount of fresh feces of ABX chicken and SPF chicken, SSL buffer was added and mixed thoroughly, and DNA was extracted from intestinal bacteria using Hi-Pure Stool DNA Kits (Qiagen). Amplification with a PCR kit amplified the V3+V4 region of the 16S rRNA gene. The PCR products were purified using the AMPure XP Beads kit (Qiagen), quantified using the ABI Step One Plus Real-Time PCR System, and then pooled and sequenced in the PE250 mode of the Hiseq2500 system. QIIME (version 2) and SPSS software (version 23.0) were used for alpha diversity analysis of duck intestinal flora, and beta diversity analysis was performed using Muscle (v3.8.31) and TreeBeST (v1.9.2) software. The RDP classifier software performs annotation classification of the intestinal flora species, and the LEfSe software analyzes the species differences in the intestinal microbiota.
Homogenization of fecal samples collected from chickens with depleted microbiota on day 7 after ABX treatment was spread on brain heart infusion (BHI) agar containing 10% sheep blood and incubated at 37 °C under anaerobic conditions for 2 days, and then incubated at 37 °C under aerobic conditions for 1 day to confirm effective microbial consumption.