Beyofast sybr green qpcr mix

Extraction of total RNA from liver tissues (up to 30 mg) was conducted using GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) based on the instructions afforded by the manufacturer. OD260/OD280 and OD260/OD230 of the RNA samples were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) to monitor the purity of the samples, and 28S/18S and RNA integrity number (RIN) of the RNA samples were detected with an Agilent 2100 Bioanalyzer (Agilent, USA) to evaluate the integrity of the samples. Then, the extracted total RNA with qualified purity and integrity was programmed to synthesize cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer’s protocols. BeyoFast™ SYBR Green qPCR Mix (Beyotime)-based RT-PCR was performed for quantificational assessment of the specific gene expressions in the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The mRNA expressions of specific genes were calculated based on the 2^−ΔΔCt method and standardized to β-actin expression as the internal housekeeping control. The primer sequences for amplification of specific gene are listed in Table 1.

Total RNA was extracted using Trizol reagent (Beyotime, R0016) and the ratio of A260/A280 was examined by a NanoDrop 2000 spectrophotometer (Thermo Scientific). A total of 0.5 ~ 1 µg of purified RNA was reversed-transcribed to cDNA with the BeyoRT™ II First Strand cDNA Synthesis Kit with gDNA Eraser (Beyotime, D7170S) in the following conditions: 65 °C for 5 min, 42 °C for 60 min and 80 °C for 10 min, and then subjected to real-time PCR analysis. Real-time PCR was performed using the BeyoFast™ SYBR Green qPCR Mix (Beyotime, D7260) on an ABI-7900 instrument (Applied Biosystems). The real-time PCR program steps were as follows: 95 °C for 5 min, then 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The 2^-ΔΔCt method was used to calculate relative mRNA amounts of target genes to the endogenous GAPDH control. The sequences of real-time PCR primers were listed in Table S1.

Total RNA was extracted from cultured cells using Trizol reagent (TaKaRa, Japan) as per the manufacturer's protocol. After quantifying the RNA concentration, a cDNA library was constructed using a Reverse Transcriptase kit (TaKaRa, Japan) following to the manufacturer's instructions. RT-PCR was performed on a RT PCR system (7500; ABI, USA) using BeyoFast SYBR Green qPCR Mix (Beyotime, China). Samples were normalized to internal control GAPDH. Primer sequences are listed as follows: IL-1α (forward primers 5′-AGG CTG CAT GGA TCA ATC TGT GTC-3′; reverse primers 5′-CTT CCT CTG AGT CAT TGG CGA TGG-3′), IL-1β (forward primers 5′-CTG AAA GCT CTC CAC CTC CA-3′; reverse primers 5′-TCA TCT TTC AAC ACG CAG GA-3′), IL-6 (forward primers 5′-GGT GTT GCC TGC TGC CTT CC-3′; reverse primers 5′-AGA TGC CGT CGA GGA TGT ACC G-3′), IL-8 (forward primers 5′-TCT CTT GGC AGC CTT CCT GA-3′; reverse primers 5′-TTT CTG TGT TGG CGC AGT GT-3′), MMP-2 (forward primers 5′-GCC TCT CCT GAC ATT GAC CTT GG-3′; reverse primers 5′-CAC CAC GGA TCT GAG CGA TGC-3′), MMP-3 (forward primers 5′-GCC AGG GAT TAA TGG AGA TG-3′; reverse primers 5′-ATT TCA TGA GCA GCA ACG AG-3′), and GAPDH (forward primers 5′-TCG ACA GTC AGC CGC ATC TTC TTT-3′; reverse primers 5′-ACC AAA TCC GTT GAC TCC GAC CTT-3′).

Total RNA of the frozen kidney tissue samples was extracted with Trizol reagent (Beyotime, China). The extracted RNA was qualitatively measured in NanoDrop One Microvolume UV–Vis Spectrophotometer (ThermoFisher, MA, USA). The extracted RNA was retro transcribed to complementary DNA (cDNA) by using an RNA-to-cDNA kit (Beyotime, China). RT-PCR was performed on a CFX Connect Real-Time PCR Detection system (Bio-Rad) by using the BeyoFast™ SYBR Green qPCR Mix (Beyotime, China). For quantitative results, the mRNA levels are expressed as a fold change by the 2^−ΔΔCt method using GAPDH as an endogenous control. The list of primers used in this study is provided in Additional file 1: Table S1.

Total RNA was isolated using TRIzol reagent (Beyotime, Shanghai, China) and quantified on a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription experiment was conducted using PrimeScript™ RT reagent Kit (Takara, Dalian, China) or TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Next, the BeyoFast™ SYBR Green qPCR Mix (Beyotime) was utilized to conduct qRT-PCR. The relative expression was estimated using the 2^−ΔΔCt method with U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference. The sequences of primers were: circ_LDLR: (F: 5′-GTGAGGGCTCTGTCCATTGT-3′ and R: 5′-GGTGGTCCTCTCACACCAGT-3′); LDLR: (F: 5′-CAATGTCTCACCAAGCTCTG-3′ and R: 5′-TCTGTCTCGAGGGGTAGCTG-3′); miR-195-5p: (F: 5′-GGGGTAGCAGCACAGAAAT-3′ and R: 5′-TCCAGTGCGTGTCGTGGA-3′); LIPH: (F: 5′-CTGATGCTCTACACAAGGA-3′ and R: 5′-ATGGACAATGAAGGTGGTT-3′); GALNT7: (F: 5′-GGTACCATGGCCTCATGTTG-3′ and R: 5′-GCCACCACACTGCCATATCT-3′); PSD3: (F: 5′-GCTCTGTACAACTCAATCAAGAATG-3′ and R: 5′-CCAATACGACTGATGGTCTTTG-3′); ITGA2: (F: 5′-CCTACAATGTTGGTCTCCCAGA-3′ and R: 5′-AGTAACCAGTTGCCTTTTGGATT-3′); GAPDH: (F: 5′-CTGGGCTACACTGAGCACC-3′ and R: 5′-AAGTGGTCGTTGAGGGCAATG-3′); U6: (F: 5′-TTATGGGTCCTAGCCTGAC-3′ and R: 5′-CACTATTGCGGGTCTGC-3′).