Biorad cfx thermocycler

In the populations of whole peritoneal cells or adherent PECs, real-time PCR (RT-PCR) was applied to determine the relative quantities of mRNA. The expression of genes for cytokines IFN-γ, TNF-α, IL-12p40, IL-6, IL-4, TGF-β, and IL-10, transcription factor NF-κB, and macrophage markers arginase 1, FIZZ-1, Ym-1, and iNOS was evaluated in total PECs. Adherent macrophages/monocytes were analyzed for mRNA abundance of transcription factors STAT1, STAT3, and STAT6, IFN-γ receptor (IFN-γR), IL-12p40, and IL-10. Moreover, mRNAs isolated from adherent PECs from infected mice after cultivation with ABZ, ABZ-SO, and DLE were examined for the expression of IFN-γ, IL-12p40, IL-10, arginase 1, iNOS, NF-κB, and IFN-γ receptor as previously described [28 (link)]. Total RNA was extracted from the cell using RiboZol reagent (VWR Life Science, Radnor Corporate Center, Radnor, PA, USA). Quantitative PCR analysis of the relative abundance of mRNA species was determined using the iTaq SYBR green master mix (BioRad, Hercules, CA, USA) on a BioRad CFX thermocycler (BioRad, Hercules, CA, USA). Relative mRNA expression was calculated by comparative quantification cycle (Cq), normalized to housekeeping gene β-actin utilizing the 2^−∆∆Ct method. The list of primers and their sequences are shown in Table S1 (Supplementary Materials).

The mRNA was isolated from CYP2E1 and control stable clones of HepG2 cells using the Qiagen RNeasy kit, following the manufacturer’s protocol. The mRNA was immediately reverse transcribed to cDNA using the Superscript III First-Strand Synthesis kit for complementary cDNA synthesis. The cDNA was used to quantify CYP2E1 and GFP gene expressions, and β-actin was used as the reference gene using real-time PCR. Briefly, a reaction mixture was prepared by combining 2 μL of cDNA, 2 μL of forward primer (200 nM), 2 μL of reverse primer (200 nM), 4 μL of UltraPure™ DEPC-treated water, and 10 μL of 2× SYBR^® Green SuperMix to have a total volume of 20 μL. In a thermocycler (Biorad CFX thermocycler; Bio-Rad Laboratories, Hercules, CA, USA), the mixture was run at a universal cycle, which included incubation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 55 °C for 60 s. The gene expression of GFP or CYP2E1 was calculated using the ΔCT method:
where CT refers to the threshold cycle. The fold change in the CYP2E1 expression in the CYP2E1-overexpressed cells relative to the wild-type CYP2E1 expression was estimated by the ΔΔCT method:

where ΔCT (Control Clone) is the geometric average of CYP2E1 ΔCT for all 6 control clones.