Hiscript rt supermix kit

Total RNA from hGMSCs, which were treated with uninduced, osteogenic induced, or osteogenic induced with SB431542 medium, was extracted with the trizol reagent kit (Invitrogen, Carlsbad, CA, USA). And cDNA was synthesis with Hiscript RT supermix kit (Vazyme; Nanjing, China) according to the manufacturer’s instructions. cDNA amplification was used by SYBR qPCR master mix (Vazyme), and qRT-PCR was performed by ABI QuantStudio™ 6 Flex system (Thermo Fisher Scientific, MASS, USA). All qRT-PCR reactions were performed in triplicate in each experiment. The BMPs genes (BMP2 and BMP4) forward and reverse primers are listed in Additional file 1: Table S1. The results were analyzed using the 2^−ΔΔct method and normalized by β-ACTIN.

In short, first of all, total RNA was extracted using the RNAiso Plus reagent from HK-2 cells that had been subjected to TGF-β1 or not, in accordance with the kit’s instructions (TaKaRa, Dalian, China). Secondly, using the HIScript RT SuperMix kit (Vazyme, Nanjing, China), cDNA was obtained. Afterwards, SYBR Green reagent (Vazyme, Nanjing, China) was used to conduct the RT-qPCR experiment following the kit’s instructions. Every sample was analyzed utilizing the 2-ΔΔCT value method. The specific primers were synthesized by Sangon (Shanghai, China). The reference used in this experiment was β-actin.

Total RNAs from mouse kidneys or human renal tubular epithelial HK2 cells were isolated using a Total RNA Extraction kit (R401‐01; Vazyme) according to the manufacturer's instructions. Equal amounts of mRNA were reversely transcribed to cDNA using a HiScript RT SuperMix kit (R122‐01; Vazyme). Quantitative real‐time PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Q711‐02; Vazyme) on a Viia 7 quantitative real‐time PCR instrument (Thermo‐Fisher Scientific). The primer sequences for TNF‐α (Mouse gene Tnfa, mTnfaF and mTnfaR; human gene TNFA, hTNFAF, and hTNFAR), IL‐6 (mouse gene Il6, mIl6F, and mIl6R; human IL6, hIL6F, and hIL6R), and the control GAPDH (mouse gene Gapdh, mGapdhF, and mGapdhR; human gene GAPDH, hGAPDHF, and hGAPDHR) were listed in the Table 1. For each detection, a 20 μl of reaction volume included 10 μl of master mixture, 2 μl of diluted cDNA, 0.6 μl each primer, and sterile distilled water. The mRNA levels were calculated using the 2‐ΔΔCt method and expressed as relative fold changes.