Power sybr green pcr master mix kit

Genomic mRNA was collected from 3T3L1 cells which had been incubated with or without GYY4137, NaHS, AOAA or PAG for 48 h. Cultured cells were washed with ice cold PBS and incubated with TRIzol^@ reagent (Invitrogen, Carlsbad, CA) for 1 min. RNA was extracted using Aurum Total RNA Mini Kits (Bio-Rad, Hercules, CA) and 1 μg of total RNA was transcribed into cDNA using an iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer’s protocols. Relative quantitative real-time PCR was performed by administering 3 μl of cDNA, 2 μl of primers to 5 μl of the reaction mix buffer from the Power SYBR Green PCR master mix kit (Life Technologies, Paisley, UK), the amplification reaction was monitored using a ViiA7 qPCR thermal cycler (Applied Biosystem, Paisley, UK). Expression values were determined by 2^-ΔΔCT equation and normalized with 18S housekeeping gene. The specific primers for representative genes are listed in Table 1.

The number of mononuclear cells isolated from spleen samples was standardized to 5 × 10⁶ and used for RNA isolation performed with a NucleoSpin RNA kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s protocol. RNA quality was evaluated in a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Concentrations of eluted RNA were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the samples were stored at −80 °C until further analysis.
Reverse transcription was carried out with a High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The concentration of RNA was standardized to 0.5 µg per sample. The expression of the gene encoding IFN-γ was determined with the qPCR method described previously [37 (link)] using a Power SYBR^® Green PCR Master Mix kit (Life Technologies, Carlsbad, CA, USA) and LightCycler 96 (Roche, Basel, Switzerland). The relative expression was calculated using the ΔCq method [38 (link)] normalized to efficiency corrections, average qPCR repeats and reference gene coding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in GenEx v. 6.1.0.757 data analysis software (MultiD, Göteborg, Sweden).

Total RNA was extracted from lung and spleen tissues using TRIzol™ reagent (Invitrogen) to analyze the relative gene expression. Then, PrimeSript™ first strand cDNA Synthesis Kit (Takara Bio, Tokyo, Japan) was used for the reverse transcription of 1 μg of RNA to 20 μL of cDNA, according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed using the obtained cDNA as a template with the Power^® SYBR Green PCR Master Mix kit (Life Technologies Co. Ltd., Woolston, UK). The relative expression levels of TNF-α, IL-6, IL-1β, IL-8, TGF-β, and IL-10, were calculated by the 2^{(−∆∆Ct)} method with normalization to β-actin. The specific primer sequences used in this study were: TNF-α, 5′-CCC CAA AGG GAT GAG AAG TT-3′ (forward) and 5′-CAC TTG GTG GTT TGC TAC GA-3′ (reverse); IL-6, 5′-CCG GAG AGG AGA CTT CAC AG-3′ (forward) and 5′-CAG AAT TGC CAT TGC ACA AC-3′ (reverse); IL-1β, 5′-TCG CAG CAG CAC ATC AAC AAG-3′ (forward) and 5′-CAT GTC CTC ATC CTG GAA G-3′ (reverse); IL-8, 5′-CCC GCG TTA GTC TGG TGT AT-3′ (forward) and 5′-AAC AGC CCA TAG TGG AGT GG-3′ (reverse); TGF-β, 5′-TTG CTT CAG CTC CAC AGA GA-3′ (forward) and 5′-TGG TTG TAG AGG GCA AGG AC-3′ (reverse); IL-10, 5′-ATG CAG GAC TTT AAG GGT TAC TTG-3′ (forward) and 5′-AGA CAC CTT GGT CTT GGA GCT TA-3′ (reverse); β-actin, 5′-AGC CAT GTA CGT AGC CAT CC (forward) and 5′-CTC TCA GCT GTG GTG GTG AA-3′ (reverse).

The number of mononuclear cells isolated from spleen samples was standardized to 5 × 10⁶ and used for RNA isolation. Genomic RNA was isolated using a commercial reagent kit (RNeasy Mini Kit; Qiagen, Hilden, Germany). Reverse transcription was conducted with the commercial kit (High-Capacity cDNA Reverse Transcription Kit; Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s guidelines. The relative expression of the genes encoding CD4 and CD8 T cell receptors and IFN-γ was determined as described previously [22 (link)], using a commercial reagent kit (Power SYBR^® Green PCR Master Mix kit; Life Technologies, Carslbad, CA, USA) and a LightCycler 96 (Roche, Basel, Switzerland). The relative expression was calculated using the 2^−ΔΔCq method [35 (link)] normalized to efficiency corrections, average RT and qPCR repeats, control group (H), and reference gene coding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in GenEx v. 6.1.0.757 data analysis software (MultiD Analyses, Göteborg, Sweden).

Total liver RNA was isolated using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized from 2 μg total RNA using Superscript III reverse transcriptase (Invitrogen) primed by oligo (dt)_12-18 (Invitrogen) and random primers (Invitrogen). Real-time qPCR was performed with Power SYBR® Green PCR Master Mix kit (Life Technologies) using the StepOnePlus-Real time PCR System (Applied Biosystems). Data were analyzed with the StepOne software (Applied Biosystems). Standard curves were used to calculate mRNA abundance relative to that of a control gene, cyclophilin. Real-time qPCR primers are summarized in supplemental Table S1. All primers used to assess expression of liver carboxylesterases are listed in supplemental Table S2. All primers were synthesized by Integrated DNA Technologies.