Abi q6 real time pcr system

To identify key miRNAs associated with SC treatment in asthma, three exosomal miRNAs were selected for real-time PCR in six asthma mice and six SC-treated asthma mice, based on high their changed fold and abundance. RNA was isolated from BALF-derived exosomes of six asthma model mice and six SC treatment mice using TRIzol (Invitrogen, Carlsbad, CA, USA). Synthesis of cDNA was performed by specific reverse primer using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, CA, USA). Real-time PCR was performed using SYBR Green Real-time PCR Master Mix (TOYOBO, No. QPK-201) and run on an ABI Q6 Real-time PCR System (Applied Biosystems Inc, USA). All reactions were performed in triplicate. The PCR process was as follows: one cycle of 95°C for 10 min, then 40 cycles of 95°C for 5 s, and annealing and extension at 55–58°C for 30 s. Data were analyzed using the 2^−ΔΔCt method. U6 was used as the reference genes, and primer sequences are shown in of the Supplementary Materials.

Total RNA from ileum tissue was extracted using Trizol reagent (Servicebio, Wuhan, China), and then reverse transcribed with a PrimeScript RT Master Mix Kit (TaKaRa Bioengineering Inc., Beijing, China) according to a standard technique to synthesize cDNA. The quantitative analysis of real-time PCR was conducted in triplicate and carried out on an ABI Q6 Real-Time PCR system (Applied Biosystems, Inc., Carlsbad, CA, USA) using the SYBR Green qPCR Master Mix (High ROX) (Servicebio, Wuhan, China). The primer sequences of GAPDH, ZO-1, Occludin, Reg3β and Reg3γ are listed in Table 2. The results were normalized to GAPDH expression and calculated using the 2^−△△Ct method.

Total RNA samples were isolated from strawberry shoot apices using the E.Z.N.A. Total RNA Kit (Omega, R6834-01, Norcross, GA, USA) according to the manufacturer’s protocol. First-strand cDNAs were synthesized from total RNA using HIScript II Reverse Transcriptase (Vazyme, R233-01, Nanjing, China). The qRT-PCR reactions were performed in 10 μL volumes containing 1 μL cDNA as a template using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02, Nanjing China) on an ABI Q6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCT method was used to analyze the qRT-PCR expression data [72 (link)]. FaACTIN was used as the internal control for the normalization of gene expression in strawberries. The name and sequences of gene-specific primers are given in Table S1.