Rt qpcr system

Total RNA was extracted using the Hot borate method described by Wan and Wilkins [97 (link)], the cDNA strand was synthesized with the HiScriptII Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Cat No. R223-01). RT-qPCR was performed with GoTaq® qPCR and RT-qPCR Systems (Promega Cat No. A6001) using a Light Cycler 480 Real-Time PCR Detection System (Roche, Rotkreuz, Switzerland). Primers of LcGRASs, and two reference genes GAPDH and EF [98 (link)] were designed by Primer Premier 5.0 (Additional file 1: Table S7). Each expression profile was independently verified in three biological replicates. The relative expression level of each gene was calculated by the 2^-△△Ct method [99 (link)].

The total RNA of yak tissue was extracted using the Trizol method, and the integrity of RNA was identified with 1% agarose gel electrophoresis. cDNA was synthesized via reverse transcription using a translator first-strand cDNA synthesis kit (Roche, Shanghai, China) and stored at −20 °C for further analysis. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using Go Taq^®qPCR and RT-qPCR Systems (Promega, Madison, WI, USA) to investigate the expression levels of the GHR messenger RNA (mRNA) in each tissue. Three replicates were selected for each sample. The primer information is provided in Supplementary Table S1. The RT-qPCR conditions were as follows: 95 °C for 3 min, 95 °C for 5 s, 64 °C for 20 s for a total of 40 cycles, and 72 °C for 30 s. The results were analyzed using the 2^−ΔΔCT method [20 (link)].

Total viral RNA from a culture supernatant and/or monolayers was extracted using QIAamp Viral RNA (Qiagen®) according to the manufacturer’s instructions. Quantitative RT‒PCR was performed using GoTaq® Probe qPCR and RT-qPCR Systems (Promega) in a StepOne™ Real-Time PCR System (Thermo Fisher Scientific) ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Amplifications were carried out in 25 µL reaction mixtures containing 2× reaction mix buffer, 50 µM of each primer, 10 µM of probe, and 5 µL of RNA template. Primers, probes, and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect SARS-CoV-2⁵² . The standard curve method was employed for virus quantification. For reference to the cell amounts used, the housekeeping gene RNAse P (F-5′-AGATTTGGACCTGCGAGCG-3′, R-5′GAGCGGCTGTCTCCACAAGT-3′, P-5′-FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ-1-3′) was amplified. The Ct values for this target were compared to those obtained for different cell amounts, 10⁷ to 10², for calibration. Alternatively, genomic (ORF1; F-5′-ATGAGCTTAGTCCTGTTG-3′, R-5′-CTCCCTTTGTTGTGTTGT-3′, P–5′-FAM-AGATGTCTTGTGCTGCCGGTA-BHQ-1-3′) and subgenomic (ORFE; F-5′ACAGGTACGTTAATAGTTAATAGCGT-3′, R-5′-ATATTGCAGCAGTACGCACACA-3′, P-5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ-1-3′) were detected, as described elsewhere^{53 (link)}.