Transcriptor first strand cdna synthesis kit

Total RNA was extracted from cartilage tissue and BMSCs with TRIzol reagent (Life Technologies, USA). The concentration and purity of extracted RNA were detected by a spectrophotometer (Thermo, USA), and finally the RNA concentration was modulated to 1 μg/μl. Then, the total RNA was reverse-transcribed into cDNA with the help of a Transcriptor First Strand cDNA Synthesis Kit (Vazyme, China) according to the manufacturer's enchiridion. Subsequently, the cDNA was mixed with SYBR Green Supermix (Servicebio, China) and the primers (Table 1) utilizing the ABI Step One RT-PCR thermal cycler (ABI StepOne, USA) in a 10 μl reaction mixture for real-time quantitative PCR (RT-qPCR). In the end, the 2^-ΔΔCt method was employed to determine relative mRNA expression level, and the GAPDH gene was used as a reference value to normalize.

Total RNA was extracted using TRIzol (Cat# T9424; Sigma Aldrich, St Louis, MO, United States). RNA was reverse-transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Cat# R323-01; Vazyme Biotech Co., Nanjing, China) according to the manufacturer’s protocol. RT-qPCR analysis was performed using a ChamQ SYBR Master Mix (Cat# Q311-03; Vazyme Biotech Co) and a LightCycler 480 QPCR System (Roche Holding AG). The results were normalized to the internal control β-actin. The primers used are shown in Table 2.

Infected N2a cells were harvested at 24 h post infection (hpi) and total RNA was prepared using TRizol reagent (Magen, Guangzhou, China) following the manufacturer’s instructions. Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). Each reaction was performed using SYBR Green Master Mix (Vazyme Biotech). Quantitative real-time PCR (qRT-PCR) was performed using a CFX connect Real-Time System (Bio-Rad, Hercules, CA, USA). The levels of AAK1 mRNA, VSV N mRNA, and RABV genomic RNA (gRNA) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers for amplification of AAK1 were as follows: forward 5′-AGTTTGCCCCCATAGCACTC-3′ and reverse primer 5′-CCTAGAGTGCCCACCTTGTG-3′. The sequences of the primers for amplification of VSV N mRNA were as follows: forward 5′-ACATATGGGAGAATGATCC-3′ and reverse primer 5′-TCTTGAGACTATGGTTCCG-3′. The sequences of the primers used for amplification of gRNA and GAPDH were as described previously [31 (link)].

Treated cells were harvested and total RNA was extracted using TRizol reagent (Magen, Guangzhou, China) according to the manufacturer’s protocol. Reverse transcription (RT) was performed using the Transcriptor First Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). Each reaction was performed in triplicate using SYBR Green Master Mix (Vazyme Biotech). Quantitative real-time PCR (RT-qPCR) was performed in a CFX connect Real-Time System (Bio-Rad, Hercules, CA, USA). The levels of TRIM21 mRNA, IFNα mRNA, IFNβ mRNA, and RABV genomic RNA (gRNA) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences of TRIM21 were used: forward, 5′-GCTCCCTCATTTACACCTTCTCG-3′, reverse, 5′-GGCTCCTGACCATCACATCTTTAG-3′. The primer sequences of IFNα, IFNβ, gRNA, and GAPDH were described previously [60 (link)].

The mRNA expression levels of the major tight junction (TJ) protein ZO-1, Claudin1, Occludin, and mucoprotein mucin (Muc) 2 were investigated by RT-qPCR. Total RNA was obtained from ileac tissues by the RNAiso Plus kit, and cDNA was generated by the Transcriptor First Strand cDNA Synthesis kit (Vazyme, Nanjing, China). The PCR reactions were performed by Stormstar SybrGreen qPCR Master Mix (Promega, Madison, USA) on the Go Taq^® SYBR-Green qPCR Master Mix (Promega, Madison, USA). Especially, in this study, the β-actin was used as the internal reference gene (Supplementary Table 2.3).