Mirvana mirna isolation kit

miR-200a was isolated from cells or homogenized liver tissues using miRVana miRNA isolation kit (Takara, Dalian, China). Real time PCR assays were performed using PrimeScript^TM RT reagent Kits (TaKaRa), SYBR Green^® miRcute miRNA Realtime PCR Kit (Tiangen, Beijing, China), SYBR Green^® Realtime PCR Master Mix and Permix Ex Taq (TaKaRa) according to the manufacturer’s instructions.
Primers used were as follows: rat β-catenin, (Forward) 5′-CTT ACG GCA ATC AGG AAA GC-3′ and (Reverse) 5′-GAC AGA CAG CAC CTT CAG C-3′; GAPDH, (Forward) 5′-CGG ATT TGG TCG TAT TG-3′ and (Reverse) 5′-GAA GAT GGT GAT GGG ATT-3′. Primers for U6 and miR-200a were purchased from Sangon Biotech (Shanghai, China). The relative gene expression was normalized to the level of GAPDH while expression of miR-200a was normalized to the level of U6. All reactions were performed in triplicates for each sample. At least three independent experiments were carried out for each experimental condition.

Total RNA plus miRNAs were isolated from cultured cells or tissue using the mirVana miRNA isolation kit (TaKaRa, Dalian, China), reverse transcribed, and then subjected to SYBR Green-based RT-PCR analysis (TaKaRa). qRT-PCR assays for miR-200a and β-catenin were performed using the PrimeScript RT Reagent Kit (TaKaRa), SYBR Green miRcute miRNA Real-Time PCR Kit (QIAGEN, Hilden, Germany), SYBR Green Real-Time PCR Master Mix, and Premix Ex Taq (TaKaRa) according to the manufacturers' protocols.
The assays were performed with the following primers: miR-200a 5′-CGT AAC ACT GTC TGG TAA CGA TGT-3′; U6 5′-CGC AAG GAT GAC ACG CAA ATT CGT-3′; β-catenin forward 5′-GAA ACG GCT TTC AGT TGA GC-3′ and reverse 5′-CTG GCC ATA TCC ACC AGA GT-3′; and GAPDH forward 5′-CGG ATT TGG TCG TAT TG-3′ and reverse 5′-GAA GAT GGT GAT GGG ATT-3. These primers were purchased from Sangon Biotech (Shanghai, China). miR-200a level was normalized to U6, and β-catenin level was normalized to GAPDH. Gene expression was analyzed using the 2^−ΔΔCT method. At least three independent experiments were conducted for each experimental condition.

The isolated exosomes from the plasma of T and C groups were submitted to Yanzai Biotechnology (Shanghai) Co. Ltd (Shanghai, China) for small RNA sequencing. A mirVANA miRNA Isolation kit (Takara, Beijing, China) was used to extracted total RNA from the exosomes. After determining the concentration and quality, the total RNA was sequenced using Illumina Miseq platform. After data pre-processing, clean data were aligned to the Rfam database, Repbase database and miRbase database to annotate miRNAs using bowtie software. After that, differential expressed miRNAs (DE-miRNAs) were identified with the thresholds of |log₂Fold change (FC)| > 1.5, as well as P value < 0.05. Then, the targets of the identified DE-miRNAs were forecasted by miranda, as well as were subjected for functional analyses based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. with P value < 0.05 was set as the standard of the significant enrichment.