Sybr green

The total RNA was extracted from cells using an RNA Extraction Kit (Accurate Biotechnology, Danyang, China). Total RNA was reverse-transcribed to cDNA using a one-step RT-PCR kit (Accurate Biotechnology, China). RT-qPCR was performed using SYBR Green (Accurate Biotechnology, China) in an RT-PCR machine (Agilent Technologies, Santa Clara, CA, USA). Values were normalized to GAPDH mRNA levels. The gene primer sequences used for qRT-PCR are listed in Table 1.

Trizol Reagent (Invitrogen) was utilized for total RNA extraction from BUMPT cells, HK-2 cells, and kidneys of C57BL/6 J mice. Next, approximately 40 ng of total RNA was reverse transcribed to cDNA using the Prime-Script-RT-Reagent-Kit and the gDNA-Eraser-Kit (RR047A; TaKaRa, Japan). qRT-PCR and SYBR Green (AG11728; Accurate Biotechnology (HUNAN) CO., LTD, ChangSha China) were employed to detect the expression levels of circRNA, micoRNA, and target gene mRNA. Relative quantification was performed by Roche LC 480 determination of ΔCt values (F. Hoffmann-La Roche, Ltd.).
Primers:
Circ_26986 (F: GAACGAACTGCACTCCGCTCTC, R: GCTGCTGGCTTGTCTTGATGATTG)
miRNA-29b-1-5P (F: GCACCGTGCTGGTTTCATATGG, R: ATCCAGTGCAGGGTCCGAGG
RT primer: ATCCAGTGCAGGGTCCGAGG)
hsa_Circ_0072463 (F: TTCCGATGACCAGTTACACAA, R: TTGGTAGTAGCGGCTCCAGT)
PAK7 (F: CTGGGAGAGGTTTGGGAGGAGAG, R: AGGGAACTACTACGGCTGGGAAG)
U6 (F: AGAGAAGATTAGCATGGCCCCTG, R: CAGTGCAGGGTCCGAGGT)
Endogenous Reference Human (F: CCTGGCACCCAGCACAAT, R: GGGCCGGACTCGTCATA)
Endogenous Reference Mouse (F: GGCTGTATTCCCCTCCATCG, R: CCAGTTGGTAACAATGCCATGT)

Total RNA was isolated from mouse liver using TRIzol reagent (Tsingke Biotech, Xian, China). 2 μg total RNA was then used for reverse transcription reaction using the cDNA synthesis kit (Deeyee, China). SYBR Green (Accurate Biotechnology, Hunan, Changsha, China) was used for quantitative real-time PCR. Each sample was analyzed with GAPDH as the internal control. The quantification of mRNA expression was calculated using the (2^−ΔΔCt) method. The primer pairs used in this study were listed in Supplementary Table S2.

RNA was extracted using an RNA extraction kit (Takara Bio), and subsequently reverse transcribed into cDNA using Evo M-MLV RT Premix (Accurate Biotechnology). The reaction conditions were 37°C for 15 min, 85°C for 5 s, and 4°C for 10 min, and a 10-μL solution containing cDNA (2 μL), ddH₂O (2 μL), primers (1 μL), and SYBR Green (5 μL; Accurate Biotechnology) was prepared for RT-PCR according to the manufacturer’s instructions. The sequences of the primers used are provided in Table 1. GAPDH was used as an internal control, and the data were calculated by the 2^–ΔΔCT method.

Total RNA of BMDMs was extracted using RNA Purification kit (B0004DP, EZBioscience) according to manufacturer instructions. Reverse transcription into cDNA was performed using Evo Moloney Murine Leukemia Virus RT Premix (AG11706,Accurate Biology). Quantitative real-time PCR was performed using SYBR Green (AG11702, Accurate Biology) on QuantStudio5 (Applied Biosystems, USA) according to manufacturer protocol. The PCR primers are shown in (Table S2). The Beta-Actin genes were used as internal control. The relative amount of each gene was calculated using the 2^-ΔΔCT method.

Six genes (cyp11a1, star, lhcgr, hsd17b3, itr, and cxcl12) were chosen for validation of the DEG data using quantitative real-time PCR (RT-qPCR). The experiments used SYBR Green (Accurate Biotechnology Co. Ltd., Changsha, Hunan, China) detection in a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). The 18S rRNA gene was used as the reference gene in these experiments. An analysis of relative gene expression data was conducted using the 2^−ΔΔCt method. All reactions were performed in triplicate.