METHODS AND MATERIALS

ERDC TN-DOER-R4

September 2004

major contaminant classes such as PAHs, PCBs, and 2,3,7,8 tetrachlorodibenzo[p]dioxin

(2,3,7,8 TCDD).

METHODS AND MATERIALS:

Cell Exposures and cDNA Array Assay. HepG2 cells were exposed to the model chemical

contaminants, and the cellular mRNA was harvested. For each exposure, four replicate T-150

culture flasks were seeded with 5 x 106 HepG2 cells, allowed to grow to 80-percent confluency,

and then dosed. Table 1 lists nominal dose levels and concentrations of the individual chemicals

in the mixtures. Contaminants were added in 150-uL aliquots of isooctane to the 15-mL

minimum essential medium (MEM) media in each flask; a solvent control (150 uL of iso-octane)

was dosed at the same time. Cells were exposed for 16 hr to either pure 2,3,7,8-

tetrachlorodibenzo[p] dioxin (TCDD, Ultra Scientific #RPE-029S), a PAH mixture (Ultra

Scientific #PM-810), or a PCB mixture (Aroclor 1254, EPA-Research Triangle Park #5705),

each at three dose levels. All dose levels for all compounds in this investigation were below

acutely toxic doses, as monitored by trypan blue viability stain. Cells were exposed to the test

chemicals for 16 hr, washed with 1x phosphate buffered saline (PBS), counted, and centrifuged.

The cell pellets were stored in 5 volumes of RNAlater (Ambion #7021) at -20oC for RNA

isolation. The mRNA was isolated using Clontech's AtlasTM Pure Total RNA Labeling Kit with

modifications; details of the RNA isolation procedure are provided in Appendix A.

Following isolation, the mRNA was converted into cDNA (Figure 1) using reverse transcription.

Procedures for Clontech's SpotLightTM Random Primer Labeling Kit were followed (http://www.

bdbiosciences.com/clontech/techinfo/manuals/PDF/PT3516-1.pdf). The only modification was

the use of the CDP-Star primers provided with the arrays. Briefly, the extracted mRNA was

placed in a tube with "primers," or short DNA sequences (oligonucleotides), which bind to

complementary sequences on the mRNA targets; primers are optimized for the genes targeted on

the array, and used as provided by the manufacturer with the arrays. An enzyme then binds to the

primer/mRNA complex and makes a complementary DNA copy of the mRNA sequence

(cDNA). The cDNA is also labeled during this step with biotin, which allows chemiluminescent

visualization after it is bound to the array.

The resulting cDNA was bound (hybridized) to the array in a complementary sequence-specific

manner during overnight exposure. Gene responses were quantified by the amount of biotin-

labeled cDNA bound at each spot on the array as detected via a light-producing reaction in which

Streptavidin-bound horseradish peroxidase binds specifically to the biotin label incorporated in

the cDNA. Methodologies for the Clontech SpotlightTM Chemiluminescent Hybridization and

Detection Kit were followed without modification for both hybridization and detection. The light

output was captured using a digital imaging analysis system (AlphaInnotech's Fluorchem 8000).

Representative results from control and exposed arrays are shown in Figure 2.

Data analysis. The digital images of the arrays were analyzed with AtlasImage 2.0 (Clontech),

which provides local background and spot densitometry data for all genes. Background corrected

data were summed for all genes in the array, and the data were then normalized to the total

intensity of all spots to allow comparisons between arrays, since the amount of cDNA applied to

each array may differ.