Total rna extraction kit

A hematology analyzer (Siemens, Germany, ADVIA2120i), a PCR system (ABI Company, USA, 7500), a Total RNA Extraction Kit, an EasyPure miRNA Kit, a Reverse transcription+PCR Kit, and the TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China, ER601-01, AT351-01) were used. The miR-21 primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Table 1).

Total RNA was isolated from the mid-ileal mucosa and granulosa cells using a total RNA extraction kit (TransGen Biotech Co. Ltd, Beijing, China) in accordance with the manufacturer's instructions. The quality and concentration of extracted RNA were determined by agarose gel electrophoresis and nucleic acid quantification, respectively. The cDNA was synthesized by the cDNA reverse transcription kit (Takara, Dalian, China). The obtained cDNA was used for gene expression analysis using SYBR green qPCR master mix (Takara, Dalian, China). The amplification conditions are as follows: 95 °C for 15 s followed by 40 cycles of 95 °C for 30 s and 60 °C for 34 s with a final melting curve analysis. The specific primers were designed online (https://bioinfo.ut.ee/primer3-0.4.0/) and showed in Table S3. The β-actin was used as a housekeeping gene to normalize target gene expression.

The total RNA extraction kit was purchased from Transgen Biotech Company (Beijing, China). MMLV reverse transcriptase and Taq DNA polymerase were purchased from Promega Corporation (Madison, WI, USA). PCR primers for the detection of SOCS-1, SOCS-3, NF-κB p65 and β-actin mRNA were designed using the OLIGO Primer Analysis Software, version 5.0 (NBA, Software and Research Services for Tomorrow’s Discoveries, National Biosciences, Plymouth, MN, USA) (Table 1).