Sybrs premix extap 2

In order to determine the expression level of CB2 receptors following RSV infection, total RNA was extracted from BAL cells and homogenized left lung lobe using RNX reagent (SinaClone, Iran), according to the manufacturer's instructions. Extracted mRNA was converted into cDNA using 1 µg total RNA in a 25 µl reaction volume with a high-capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) according to the manufacturer's instructions. For CB2 gene expression, Real-time RT-PCR was performed on an ABI PRISM 7900 sequence-detection system (Applied Biosystems, USA) using the SYBRs Premix ExTapTM II (Takara, Japan). The reaction conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 57°C for 30 s, and 60°C for 30 s. Melting curve conditions were as follows: 95°C for 15 s and 65°C for 1 min. The relative level of gene expression was determined by the comparative threshold cycle method as described by the manufacturer. Level of CB2 gene expression was normalized to those of the housekeeping gene (β-actin) using the 2^−ΔΔCt method and expressed in the graphs as “relative expression”. The following primer pairs were used: CB2: 5′-GGTGGACTTGTTGCCCTAGT-3′ (F), 5′-TAGAAGCCAGCCCAGTAGGT-3′ (R), and β-actin: 5′-GCTCTGGCTCCTAGCACCAT-3′ (F), 5′-GCCACCGATCCACACAGAGT-3′ (R).

Total RNA was extracted from samples using TRIzol reagent (Pufei, Shanghai, China) and reversely transcribed into cDNA using the PrimeScript RT reagent Kit (Takara Bio, China), following by PCR amplification using SYBRs Premix Ex Tap TM II (Takara Bio, Shiga, Japan) in a real-time PCR system (Bio-Rad, Hercules, CA) (Xie et al., 2021a (link); Xie et al., 2021b (link)). GraphPad Prism was used to conduct statistical analyses. The primers sequences as below: RRM2 forward-CATTGTGAGGTACAGGCGGAAG, reverse-GAAATGGTCTGAGCTGGCAGAAG, HBx forward-CCCGTCTGTGCCTTCTCATC, reverse-CCCAACTCCTCCCAGTCTTTA, ACTB forward-TGGCACCCAGCACAATGAA, reverse-CTAAGTCATAGTCCGCCTAGAAGCA. GAPDH forward-GACATCAAGAAGGTGGTGAAGCAG,reverse-GTCAAAGGTGGAGGAGTGGGT.