Mirna 1st strand cdna synthesis kit

Total RNA was extracted using TRIzol reagent (Accurate Biotechnology, Hunan, China) and converted into cDNA (Evo M-MLV RT Premix for qPCR; Accurate Biotechnology). The miRNA RNAs were converted into cDNA using a miRNA 1st strand cDNA synthesis kit (Accurate Biotechnology, Hunan, China). β-Actin and U6 were used as reference controls for mRNAs, lncRNAs, and miRNAs. Primers were designed by Accurate Biotechnology (Hunan, China) (Table S2). qPCR was performed using the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China) and the Applied Biosystems 7500 Real-Time PCR Detection System. The data were analyzed using the 2^−ΔΔCt (Livak) relative expression method. All experiments were repeated three times.

We used TRIzol reagent according to the manufacturer’s instructions to extract total RNA from growth plates. For mRNA, PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, San Jose, CA, USA) was used to convert total RNA to cDNA (37 °C for 15 min and 85 °C for 5 s) and TB Green^® Premix Ex Taq™ (Takara, San Jose, CA, USA) was used to amplify the cDNA as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 34 s. For miRNA, cDNA was synthesized using the miRNA 1st strand cDNA synthesis kit (Accurate Biotechnology Co., Ltd, Hunan, China). SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology Co., Ltd, Hunan, China) was used for the RT-qPCR assay as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 20 s. Data were normalized to GAPDH (for mRNA) and U6 (for miRNA). The primers used in these experiments are listed in Table S2 . RT-qPCR results were calculated using the 2^−ΔΔCT method (Livak & Schmittgen, 2001 (link)). All groups had three independent samples and all of the samples were conducted in triplicate.

Total RNA from mouse colonic tissues was isolated with RNAiso Plus (Takara, Dalian, China). RNA quality was evaluated using agarose gel electrophoresis. Subsequently, RNA samples were converted to cDNA using a reverse transcription kit (Takara, Dalian, China). Next, cDNA samples were examined for tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-23 (IL-23), Claudin-1, Occludin, and N-myc downstream regulated gene 1 (Ndrg1) expression. Data were normalized using β-actin as a reference gene. Primers were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov, last accessed on 1 May 2023). Primers used in the present study are listed in Table 1. To test the expression levels of miR-199a-5p and -3p, small RNA was reverse transcribed via miRNA 1st-Strand cDNA Synthesis Kit (Accurate Biotechnology, Changsha, China). Forward primer sequences for miR-199a-5p were as follows: 5′-CCCAGTGTTCAGACTACCTGTTC-3′; for miR-199a-3p: 5′-ACAGTAGTCTGCACATTGGTTA-3′. Reverse sequences and U6 (small nuclear RNA) primers were included in the kit. Real-time PCR reactions were performed on a Quanstudio 5 instrument (Thermo Fisher, Singapore). All samples were analyzed simultaneously in triplicate. Relative expression was analyzed using the 2^−ΔΔCT method.