Sybr green qpcr supermix

RNA from primary hepatocytes and liver tissue was extracted using TRIzol reagent using the manufacturer’s protocol. cDNA was synthesized using random hexamers (High-Capacity cDNA Reverse Transcription Kit, ThermoFisher-Applied Biosystems). Real-time quantitative PCR was performed using SYBR green qPCR Supermix (Biorad) and QuantStudio 6 (Applied Biosystems). Primers are provided in Supplementary Table 1. Gene expression was quantified using the ddCt method.

To further confirm the reliability of the microarray assay, qRT-PCR assays were performed. Briefly, single-stranded cDNA was synthesized using the RevertAid kit (Fermentas Life Science, Burlington, ON, Canada), according to the manufacturer's protocols, with random primers and 1 μg RNA from the same samples used in the microarray. Real-time PCR was conducted using the SYBR Green q-PCR SuperMix (Bio-Rad, USA). The primers used are listed in Table 1. Each qRT-PCR reaction included 10 μL SYBR Green q-PCR SuperMix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), and 1 μL cDNA. The total volume was adjusted to 20 μL with ddH₂O. The following thermocycler parameters were used to generate the dissociation curve: (1) 95°C for 5 min; (2) 40 cycles of 95°C for 15 s, 60°C for 40 s, and 72°C for 20 s; and (3) 65 to 95°C. The 7500 System SDS software (ABI, USA) was used for acquiring the Ct values with manual thresholds. PCR amplifications were performed in triplicate for each sample. Gene expression levels were normalized relative to the expression of GAPDH using the ΔΔCT method. The gene expression levels between the groups were compared using 2^−ΔΔCT and Student's t-test. p values < 0.05 were considered statistically significant.

For qRT-PCR, total RNAs were extracted by the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 2 μg of total RNA in a 20 μL volume using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and oligo (dT)₁₅ primers, and diluted with 80 μL distilled water. Quantitative PCR was carried out using SYBR Green qPCR SuperMix (Biorad, Hercules, CA, USA). The primers were designed using Primer Express software (Thermo Scientific, Waltham, MA, USA), and the primer sequences used for RT-PCR were listed in Table S2. Each experiment was repeated at least three times. The qPCR amplifications was performed using a Biorad CFX96 (Biorad, Hercules, CA, USA) under the following conditions: 95 °C for 30 s, followed by 39 cycles at 95 °C for 15 s, and 60 °C for 30 s.

Total RNA from undifferentiated ASCs and from ASCs that had undergone adipogenic differentiation was extracted using RNA extraction kit (Qiagen, Germantown, MD, USA) and then digested with DNase I (Qiagen)). A total of 1 μg of mRNA was used for cDNA synthesis with an Applied Bioscience purification kit (Thermo Fisher Scientific, USA). qRT-PCR was performed using the SYBR Green qPCR SuperMix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Oligonucleotide primers were designed with the vendor’s software (IDT, USA). Table 2 lists the primer sequences used for qRT-PCR. PCR conditions were: 2 min at 95 °C, and 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The target and reference genes were amplified in separate wells. All reactions were performed in duplicate. The 2^−ΔΔCT method was used to quantify gene expressions and data were normalized to GAPDH, which was used as an internal control.

Total RNA from ASCs was extracted using an RNA extraction kit (Qiagen, Germantown, MD, USA). One microgram of mRNA was used for cDNA synthesis with an Applied Bioscience purification kit (Thermo Fisher Scientific, USA). qRT-PCR was performed using the instructions from the SYBR Green qPCR SuperMix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Oligonucleotide primers were designed using the vendor’s software (IDT, Coralville, IA, USA). Table 2 lists the primer sequences used for qRT-PCR. PCR conditions: 2 min at 95 °C and 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The target and reference genes were amplified in separate wells. All reactions were performed in duplicate. The 2^{(−∆∆CT)} method was used to quantify gene expressions and normalized data to GAPDH, which was used as an internal control.

Real-time quantitative PCR was carried out with SYBR Green qPCR SuperMix (Bio-Rad) using the CFX-96 system (Bio-Rad). Total cellular RNA was isolated using TRIzol reagent, and cDNA was synthesized from 1 μg of total RNA using oligo-dT and Moloney murine leukemia virus reverse transcriptase (Toyobo, Japan). Relative expression levels of the genes were calculated using the 2^−ΔΔCT method.