7500 fast rt pcr

The mRNA level of PDE7B in testosterone treated HepG2s was determined by real-time PCR. Beta-actin by Applied Biosystems (Carlsbad, CA, USA) was chosen as an endogenous housekeeping control gene. Quantitative real-time PCR was performed using the 7500 Fast rtPCR from Applied Biosystems. Reaction mixtures contained SYBR green reaction mix from Kapa Biosystems (Woburn, MA, USA), PDE7B primers (as described in Pekkinen et al., 2008 (link)), 4 μl cDNA template in a total volume of 15 μl. Thermal cycling conditions included activation at 95°C (10 min) followed by 40 cycles each of denaturation at 95°C (15 s) and annealing/elongation at 60°C (1 min). Each reaction was performed in triplicate and no-template controls were included in each experiment. The untreated sample was employed as a calibrator and the delta CT-formula was used as described in the literature (Livak and Schmittgen, 2001 (link)). The gene expression was quantified as the yield of the target gene relative to that of Beta-actin gene.

Real-time PCR was used to quantify gene expression via the amplification of mRNA transcripts 7500 Fast RT-PCR (Applied Biosystems, Foster City, CA, USA). The cells were grown into 12-well culture plates to 70% confluency before being exposed to the NPs for 6 h at 12.5 µg/mL. Total RNA was extracted using TRIzol™ LS Reagent (ThermoFisher Scientific, Pleasanton, CA, USA) based on the manufacturer’s protocol. Jenway Genova Nano (Cole-Parmer, Vernon Hills, IL, USA) was used to measure RNA concentration and quality. Using RT Master Mix HY-K0510 (MedChemExpress, South Brunswick Township, NJ, USA) and a Veriti thermocycler (Applied Biosystems, Woburn, MA, USA), mRNA was reverse transcribed into cDNA. Real-time PCR was carried out using an SYBR Green qPCR Master Mix (Low ROX) HY-K0522 (MedChemExpress, NJ, USA), reverse and forward mouse primers (Table 2) for tumor necrosis factor-alpha (TNF-α), Interleukin-1 beta (IL-1β), and GAPDH, which were obtained from integrated DNA technology (USA). The ΔΔC_T technique was used to calculate gene expression in comparison to a housekeeping gene (GAPDH).

Taq‐CNV assay for FUBP3 gene (reporter: FAM dye, quencher: MGB–NFQ) and reference gene RNaseP (reporter: VIC, quencher: TAMRA) (Applied Biosystems, Thermo Scientific) was performed as per the manufacturer's instructions on 7500‐Fast RT‐PCR (Applied Biosystems, Thermo Scientific). Copy numbers were calculated using CopyCaller™ software (Applied Biosystems, Thermo Scientific). The copy number value of 2 indicated normal values. The values close to 1 were considered as deletions.