Abstract:

Quantitative polymerase chain reaction (qPCR) of target genes was used to test for induced gene expression in HepG2 cells treated with chrysenes and other compounds [22]. HepG2 cells were exposed to different chemicals for 6 h over a range of concentrations ranging from non-cytotoxic to modest toxicity. RNA was extracted from the cells using Qiagen’s RNeasy Plus Mini Kit (#74136) and following the manufacturer’s instructions. RNA purity and concentration were assessed using a Thermo Scientific Nanodrop 2000c spectrophotometer followed by dilution to 0.5 µg/µl in nuclease-free water to avoid RNA damage. Complementary DNA (cDNA) was synthesized using BioRad iScript cDNA synthesis kit (#170-8891) following the manufacturer’s instructions. cDNAs were stored at -20 degree C until use. Gene expression was detected using primer-probe sets to AhR-regulated genes from Applied Biosystems’ TaqMan Gene Expression Assays along with hypoxanthine phosphoribosyl transferase 1 (HPRT1) as a reference transcript. RT-PCR was conducted using a BioRad C1000 thermal cycler supplied with a CFX96 Real-Time PCR Detection System.

Suggested Citation:

Ahmad Alqassim, Charles Miller. 2019. Gene expression changes induced by methylchrysenes in HepG2 cells. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-g79v-8j14

Publications:

Alqassim, A. Y., Wilson, M. J., Wickliffe, J. K., Pangeni, D., Overton, E. B., & Miller, C. A. (2019). Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono‐methylchrysenes. Environmental Toxicology, 34(9), 992–1000. doi:10.1002/tox.22770

Purpose:

To determine the gene expression within HepG2 cells in response to chemicals.

Data Parameters and Units:

Relative fold difference (no units), concentration (nM), and chemical (the type of chemical represented by a number; 0-8). Labels for concentration: 0 = "DMSO" 1 = "B(a)P" 50 = "50nM" 500 = "500 nM' 5000 = "5000 nM" Labels for chemical: 0="Chrysene" 1="1-Methylchrysene" 2="2-Methylchrysene" 3="3-Methylchrysene" 4="4-Methylchrysene" 5="5-Methylchrysene" 6="6-Methylchrysene" 7="DMSO" 8="Benzo[a]pyrene"

Methods:

HepG2 cells were incubated in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 μg/mL streptomycin, 50 U/mL penicillin and propagated in an incubator with 5% CO2 at 37°C. The cells were seeded always at 1 x10^6 / 60 mm^2 dish for each chemical. they were left to grow for 24 h and then incubated with treatments for another 24 h. The expression levels of the target genes and reference transcript were evaluated. The levels of these amplicons in a series of chemical-treated samples were compared to the DMSO-treated sample. The comparative CT method was used to analyze the data. The averages of the comparative (CT) values for the reference gene (HPRT1) and the gene being tested in the experimental and control conditions were taken. The differences between the CT values for HPRT1 gene and tested gene were calculated as the delta comparative (ΔCT) values for the experimental and control conditions, respectively. The differences between the ΔCT values for the experimental and control conditions were calculated as the double delta comparative (ΔΔCT) values. Then, the values of 2- ΔΔCT were calculated to get the gene expression fold change.

Instruments:

BioRad C1000 thermal cycler supplied with a CFX96 Real-Time PCR Detection System.

Individual Files: