Cfx connect pcr detection system

Total RNA was isolated from CF or cardiac tissue powder using Trizol (Thermo Fisher Scientific) according to manufacturer instructions. For mRNA qPCR, reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, 4368814) and qPCR was performed using Power Up SYBR Green Master Mix (Thermo Fisher Scientific, A25779) on a Bio-Rad CFX Connect PCR detection system. Primers are listed in Supplemental Table 1. For miRNA qPCR, Taqman RT kit (Thermo Fisher Scientific, 4366596), Taqman assays (Thermo Fisher Scientific, 4427975; Assay IDs 001973, 000590, 002185), and Taqman Universal Master Mix (Thermo Fisher Scientific, 4440040) were employed.

Twenty-four hours before DNA extraction, arches were transferred from the RNA stabilization reagent into 1 mL lysis solution and stored at 4 °C. DNA was extracted from all samples (arches and swabs) using the same protocol. After vortexing, 250 µL of sample solution was combined with 250 µL lysis solution and 2 µL proteinase K (20 mg mL⁻¹). The mixture was then incubated at 37 °C for 30 min. After incubation, samples were chilled on ice for 5 min, combined with 250 L 7.5 M ammonium acetate, and vortexed for 20 s. Samples were then centrifuged at 14,000× g for 5 min and the resulting supernatant combined with 750 µL isopropanol. To facilitate precipitation, samples were then inverted for 5 min and centrifuged at 16,000× g for 10 min. The nucleic acid pellets were rinsed twice in 70% ethanol and re-suspended in 100 µL buffer (10 mM Tris, 0.05% Triton ×100).
A real-time PCR assay which distinguishes an N. perurans-specific 18S rRNA gene sequence [58 (link)] was run on all samples, in duplicate, with no-template controls. All reactions were run in a CFX Connect PCR Detection System (Bio-Rad, Hercules, CA, USA). N. perurans cell numbers were estimated based on 2880 copies cell⁻¹ as previously determined [58 (link)]. This method allowed non-lethal sampling throughout disease progression, and quantitative comparison of infection severity between treatments.

Total RNAs of decidua tissues were extracted using Trizol (Invitrogen) according to manual instructions. Quantitative RT-PCR (qRT-PCR) was performed using the HiScriptII Supermix (Vazyme, Nanjing, China) following the manufacturer’s instructions. Briefly, RNA was quantified using a Nanodrop One instrument (Thermo, MA, USA) and 1 μg was used for reverse transcription using random primers. For qRT-PCR, SYBR Green master mix and primers (final concentration at 200 nM) were used and results were analyzed in CFX Connect PCR detection system (Bio-Rad, CA, USA). Primers were designed according to a previous publication (22 (link)) for CCL8 and GAPDH, or using an open resource (www.ncbi.nlm.nih.gov/tools/primer-blast) for TRDV1, TRDV2, and TRDV3, whose sequences are listed in Supplementary Table S2. Expression levels were normalized to GAPDH and represented as fold change compared to the control (2^−ΔΔCt).

The total RNA was extracted from the intestine, liver, and kidney tissues of broilers using Trizol (Invitrogen, Carlsbad, CA, USA) according to the standard manufacturer’s instructions, and then dissolved in 50 μL RNase-free water and stored at −80 °C. The quality and concentration of RNA samples were measured by NanoDrop ND-1000 Spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). Approximately 1 μg total RNA from each sample was reversely transcribed into cDNA by TB GREEN kit (TaKaRa, Dalian, China). Quantitative RT-PCR (qRT-PCR) was performed by the CFX Connect PCR Detection System (Bio-Rad, Hercules, CA, USA). All the primers used in this study are listed in Table 5. The β-actin was used as a house-keeping gene, and the relative mRNA abundances were analyzed using the 2^−ΔΔCT method [61 (link)].

Total DNA was isolated from finely minced tissues (∼20 mg of liver, pancreas, brain and ∼15 mg of spleen) by using DNeasy 96 Blood & Tissue Kit from Qiagen, USA following the manufacturer’s guideline. Quantitive real time PCR was performed to determine the copy number of adenoviral genomic DNA in liver, pancreas, brain and spleen by using iTaq™ Universal SYBR^® Green Supermix from Bio-Rad, USA. The following primers were used based on a previous study [30 (link)]. The sequences of the forward and reverse primers were 5’-CCACCGATAGCAGTACCCTT-3’ and 5’-GACCAGTTGCTACGGTCAAA-3’, respectively. The PCR cycle consists of one cycle of 95°C for 3 min and then 40 cycles of 95°C for 10 s, 55°C for 10 s and 72°C for 30 s and one cycle of 95°C for 10 s. Purified pAdEasy vector (Agilent Technologies) with known copy number (2.2X10⁶ to 2.2X10^-1 copy) was used to prepare the standard curve for the determination of Ad viral DNA copy number in the samples. Each sample and standard were run in triplicate. A Bio-Rad CFX connect PCR detection system was used for real time PCR and data was anlyzed by Bio-Rad CFX manager. A negative control using molecular biology grade water (ThermoFisher Scientific, USA) instead of DNA template was included and the quantification cycle (Cq) values less than the negative control were considered as negative.