Hipure total rna mini kit

Total RNA was prepared with a Hipure Total RNA Mini Kit (Magen, Guangzhou, China). ReverTra Ace qPCR RT kit (TOYOBO Co., Ltd., Osaka, Japan) was used to synthesize cDNA. Quantitative PCR was carried out using the SYBR green PCR master mix kit (TOYOBO Co., Ltd., Osaka, Japan) on the StepOnePlus realtime PCR system (ThermoFisher Scientific, CA, U.S.). Quantitation of IL-6 mRNA was normalized to GAPDH by the ΔΔCt method. The primers were rat IL-6: forward: 5′-ACTTCCAGCCAGTTGCCTTCTTG-3′, reverse: 5′-TGGTCTGTTGTGGGTGGTATCCTC-3′; rat GAPDH: forward: 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′.

The total RNA was extracted from the cells according to the protocol provided by the HiPure Total RNA Mini Kit (Magen, R4111-03) and collected at 4 °C. RNA concentration was measured using NanoDrop^® ND-2000 UV–Vis spectrophotometer. And cDNA was then synthesized using ReverTrace qPCR RT Kit with 1 μg total RNA as template. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green PCR Master Mix. The thermocycling protocol was taken from a previous paper by Xiong et al. [81 (link)]. The primer sequences used in the qRT-PCR are provided in Supplementary Table S1.

Total RNA was extracted from the PP tissues and cell samples using a HiPure Total RNA Mini Kit (Magen, China). mRNA and lncRNA were reverse-transcripted from total RNA using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara, China). The RT–qPCR reaction system (in 10 µL) included 1 µL cDNA, 0.8 µL primers, 0.2 µL ROX Reference Dye, 5 μL TB Green Premix Ex Taq (TaKaRa, China), and 3 µL RNA-free water. The following PCR reaction was performed in a Quant Studio 6 Flex instrument (Thermo Fisher Scientific, United States): 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s, then a melting curve analysis (65°C–95°C). The amplification efficiencies neared 100%, and the relative expression levels of mRNA and lncRNA were calculated using the 2^-△△CT method and expressed as the fold change relative to the GF or cell control group (Livak and Schmittgen, 2001 (link)). GADPH was used to normalize the gene expression levels (Barber et al., 2005 (link); Gaur et al., 2014 (link); Li et al., 2019 (link)). The specific primer sequences in this study are listed in Supplementary Table S1-1.

Total RNA of tissues and cells was extracted using HiPure Total RNA Mini Kit (Magen). Reverse transcription was carried out with HiFi-script cDNA kit (Cwbio) in the light of the manufacturer’s instruction. The Bestar^TM Real time PCR Master Mix was applied to real-time quantitative polymerase chain reaction (RT-qPCR) by SYER Green Method. All reactions were performed on an ABI 7900HT instrument (Applied Biosystems). All experiments were implemented at three independent times and all reactions were performed in triplicate at least. The dates of RT-qPCR were normalized to U6 using the 2^−ΔΔCt method. The primers were ordered from Sangon Biotech. The mentioned primer sequences are exhibited in Supplementary Table 1.

Total RNA isolated from harvested cell lines and mouse kidney cortex using the Hipure Total RNA Mini Kit (R4111-03, Magen, China) according to the manufacturer’s instructions. RNA concentrations were determined using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). cDNA was generated with a ReverTra Ace qPCR RT kit (FSQ-101, Toyobo, Japan). RT-qPCR was performed using an ABI Step One Plus system (Applied Biosystems, USA) with SYBY Green PCR mix (QPK-201, Toyobo, Japan) in 96-well plates. The data were normalized and analyzed using the 2^−ΔΔCt method. The primers used are listed in Supplementary Table 1.