Cdna synthesis kit

Total RNA was extracted from cultured MCs and kidney glomeruli using the Trizol reagent (TaKaRa Bio, Japan). First-strand cDNAs were synthesized using a cDNA Synthesis Kit (TOYOBO, Japan) and then quantified by real-time PCR with the KOD SYBR Green qPCR Mix kit (TOYOBO, Japan) and appropriate primers. Reactions were performed on the ABI 7500FAST System (Foster City, CA). Relative gene expression levels were normalized to GAPDH expression. Primers were obtained from Sangon Biotech Co., Ltd. (Shanghai, PRC), and all primer information is available upon request.

Total RNA was isolated from the cells or SN regions using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. For the first-strand synthesis, a cDNA Synthesis Kit (Toyobo, Osaka, Japan) was used to synthesize the cDNA. All the real-time PCR were performed with SYBR Green (Toyobo, Osaka, Japan) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Inc., Hercules, CA, USA). The primers and PCR conditions for the amplification of miR-873, pre-miR-873, ABCA1, A20, GCase, cathepsin D (CTSD), NPC1 and NPC2 are listed in Table S1. Human GAPDH was used to normalize the gene expression levels, including the levels of ABCA1, A20, GCase, CTSD, NPC1 and NPC2. U6 was used to normalize the relative expression of miR-873. The gene expression levels were calculated using the 2^−ΔΔCT method relative to the internal control.

Quantitative real-time polymerase chain reaction (qRT-PCR) was applied in the study. Total ribonucleic acid (RNA) of the chondrocytes was extracted with a RNA isolation kit purchased from Omega (Guangzhou, China) based on the manufacturer’s protocol. Subsequently, a spectrophotometric microplate reader (Bio-Rad) was applied to determine the concentration of RNA molecules, and the ratio of A260/A280 was calculated to verify the purity of the total RNA. Subsequently, a cDNA synthesis kit (TOYOBO, Osaka, Japan) was utilized to reverse transcribe equal amounts of RNA (1 μg) to stable cDNA. The qRT-PCR reaction was conducted at 95 °C for 1 min, followed by 39 cycles at 95 °C for 15 s and 60 °C for 15 s. Relative expression of each target gene was normalized to that of GAPDH and analyzed using the 2-ΔΔCq method.

Total RNA was extracted from cells using Trizol reagent. RNA was reverse-transcribed using a cDNA synthesis kit (Toyobo Life Science, Japan). Real-Time PCR was performed using a StepOnePlus Real-Time PCR Detection System (Applied Biosystems Inc., CA, USA). A total volume of 20 μL reaction mixture containing 1 μL cDNA, 10 μL SYBR Green PCR Master Mix (Toyobo, Japan) and 1.6 μL each primer (10 μM) was used for this assay. GAPDH was used as reference for normalization and the relative expression of mRNA was calculated according to the ΔΔCt method.
The primer pairs used for the Real-time PCR were as follows: for the human MPST gene, the forward primer was 5′-CGGAGTCTCCTCCCTTTGGT-3′, and the reverse primer was 5′-CCTCCCTAAGATGCAGCTCG-3′. For the GAPDH gene, the forward primer was 5′-TGCCCCCATGTTCGTCA-3′, and the reverse primer was 5′-CTTGGCCAGGGGTGCTAA-3′.