Step one fast real time pcr system

RNA was extracted by Trizol® reagent (category no. 15596026, Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was then synthesized with a High-Capacity cDNA Reverse Transcription kit (category no. 4368814, Applied Biosystems, Foster City, CA, USA). High cDNA quality was checked, taking into consideration the housekeeping gene Ct values. Quantitative real-time PCR was performed in Step-One Fast Real-Time PCR system, Applied Biosystems, using the SYBR Green PCR MasterMix (category no. 4309155, Life Technologies, Monza, Italy). The specific PCR products were detected by the fluorescence of SYBR Green, the double-stranded DNA binding dye. Primers were designed using BLAST® (Basic Local Alignment Search Tool, NCBI, NIH), considering the shortest amplicon proposed and β-actin was used as the housekeeping gene. Primers were purchased by Metabion International AG (Planneg, Germany) (Table 2). The relative mRNA expression level was calculated by the threshold cycle (Ct) value of each PCR product and normalized with β-actin by using a comparative 2-ΔΔCt method.

RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA; Tibullo et al., 2013 (link)). First strand cDNA was then synthesized with Applied Biosystem (Foster City, CA, USA) reverse transcription reagent. Quantitative real-time PCR was performed in Step One Fast Real-Time PCR System Applied Biosystems using the SYBR Green PCR MasterMix (Life Technologies; Malaguarnera et al., 2011 (link); Marrazzo et al., 2011 (link)). The primer sequences used are shown in Table 1. The specific PCR products were detected by the fluorescence of SYBR Green, the double stranded DNA binding dye. The relative mRNA expression level was calculated by the threshold cycle (C_t) value of each PCR product and normalized with that of GAPDH by using comparative 2^-ΔΔCt method.

RNA was extracted from tissues by using Trizol^® reagent (Invitrogen, Cat. No. 15596026). RNA was measured, and 1 µg of RNA with a 260/280 ratio > 1.8 was used for reverse transcription by using a high-capacity cDNA kit (Applied Biosystems, Monza, Italy, Cat. No. 4368814). High cDNA quality was checked, referring to the housekeeping gene Ct values. Quantitative real-time PCR was performed in the Step-One Fast Real-Time PCR system, Applied Biosystems, using the SYBR Green PCR MasterMix (Life Technologies, Monza, Italy, Cat. No. 4309155). The specific PCR products were detected by the fluorescence of SYBR Green, the double-stranded DNA binding dye. The relative mRNA expression level was calculated by the threshold cycle (Ct) value of each PCR product and normalized with a comparative 2^−ΔΔCt method, using GAPDH as housekeeping. The sequence of primers used are listed in Table 3.

RNA was isolated using TRIzol (Life Technologies) following the manufacturer’s protocol. cDNA was generated using the transScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgen). Quantitative real-time PCR using Fast SYBR Green PCR Master Mix (Applied Biosystems) was performed on a Step-One Fast Real-time PCR System (Applied Biosystems). For RNA stability assay, the cells were plated in a 6 cm dish and incubated with actinomycin D (Santa Cruz) at 5 mg/ml for indicated time points. Total RNA samples were then isolated for qPCR analysis. The quantitative PCR primers were listed in Table S1.

Cells were treated with EFE at different concentrations (0.05, 0.1, 0.25, 0.5, 1, and 1.5 mg/mL) for 24 h and then harvested for RNA extraction using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). The Applied Biosystem (Foster City, CA, USA) reverse transcription reagent was used to obtain first-strand cDNA, and then qRT-PCR analysis was performed in a Step One Fast Real-Time PCR System Applied Biosystems using the SYBR Green PCR Master Mix (Life Technologies, Monza MB, Italy) to evaluate antioxidant gene expression (HO-1, GSR, and GCLC). Results were normalized with the housekeeping gene GAPDH using a comparative 2^−ΔΔCt method.

RNA was extracted using Trizol^® reagent (Invitrogen, Carlsbad, CA, USA) (19 ). First-strand complementary DNA (cDNA) was then synthesized with a reverse transcription reagent from Applied Biosystems (Foster City, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed in StepOne Fast Real-Time PCR System (Applied Biosystems) using the SYBR Green PCR MasterMix (Life Technologies, Monza, Italy) (20 (link)). The specific PCR products were detected with SYBR Green fluorescence. The relative messenger RNA (mRNA) expression level was calculated by the threshold cycle (C_t) value of each PCR product and normalized with that of actin using a comparative 2^−ΔΔCt method. The sequences of the primers used are presented in Table 1.

Total RNA was isolated from the cells by using Trizol reagent, and the concentrations of the extracted RNA were measured spectrophotometrically at 260 nm. RNA quality was assessed based on the absorbance ratio at 260 and 280 nm. A260/A280 values ranging from 1.9 to 2.1 were considered acceptable. The total RNA was reverse transcribed into cDNA by using a PrimeScript RT reagent kit with gDNA eraser. Real-time PCR analysis was performed using SYBR Green according to the manufacturer’s instructions. The reactions were run at 50°C for 2 min and 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min on the Applied Biosystems Step-One Fast Real-Time PCR system (Table 1). The results were quantified using 2^−ΔΔCT method.