Transcriptor cdna synthesize kit

Total RNA was extracted from the cells using the Trizol Reagent (Thermo Fisher, catalog 15596-026). cDNA was synthesized from 2 μg of total RNA in 20 μl of reaction volume using the Transcriptor cDNA Synthesize Kit (Roche, catalog 04897030001) with OligdT primer, and to detect the miR-23b-3p level the same cDNA sample was synthesized from 500 ng of total RNA with miR-23b-3p-specific stem-loop reverse transcription primer. Gene expression levels were measured by qPCR using the SYBR Green System (Roche, catalog 04913850001). Quantification of the relative expression levels was determined by ΔΔCt method. Triplicates were used in assay, and the cell experiments were repeated three times. All the primer sequences for mRNA detection are shown in Supplementary Table S2.

For RNA extraction, cartilage tissues (n = 18) from smooth and damaged articular surface of the same patients were harvested and divided into small pieces (<2 × 2 mm²). The pieces were frozen in liquid nitrogen immediately. Total RNA was isolated with Trizol reagent (Thermo Fisher, catalog 15596-026) according to the manufacturer’s protocol. cDNA was synthesized from 2 μg of total RNA in 20 μl of reaction system using the Transcriptor cDNA Synthesize Kit (Roche, catalog 04897030001) with OligdT primer for detecting mRNA expression. Meanwhile, the same cDNA sample was synthesized from 1 μg of total RNA with the mir-X miRNA First-Strand Synthesis Kits (Clontech, catalog 638315). Gene expression levels were detected by quantitative PCR (qPCR) with the SYBR Green System (Roche, catalog 04913850001). The primers for measuring mRNA are shown in Supplementary Table S2. The forward primer for miR-23b-3p detection was designed by Tiangen Biotech (Beijing, China) and reverse primer for miRNA measure was from the mir-X miRNA First-Strand Synthesis Kits. Quantification of the relative expression levels was determined by ΔΔCt method.