APPENDIX A

ERDC TN-DOER-R4

September 2004

APPENDIX A

CELL EXPOSURES: Human hepatoma HepG2 cells were counted and checked for viability

with Trypan Blue dye using a hemacytometer and microscope. Cells with >95 percent viability

or better were then seeded at ~5 x 106 per T-150 flask (150 cm2 growing surface area) and

allowed to grow for several days until the cells reached ~80 percent confluency. Replicate flasks

were then dosed with isooctane (solvent control) at 1 percent v/v or with the following

compounds at different concentrations as shown in the table below.

Table 1

Media Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD (Ultra Scientific #RPE-

029S), Polyaromatic Hydrocarbon Mix, PAH Mix (Ultra Scientific #PM-810), and Aroclor 1254

(EPA-Research Triangle Park #5705)

2,3,7,8-TCDD

PAH Mix

Aroclor 1254

Isooctane

7.5 pg/mL

1.7 ng/mL

1 ng/mL

25 pg/mL

17 ng/mL

10 ng/mL

100 pg/mL

170 ng/mL

100 ng/mL

After 16 hr of exposure to the compounds of interest, cells from the same treatment were pooled,

washed (with 1X phosphate buffered saline, PBS), counted, and centrifuged. The cell pellets

were rinsed twice with PBS before proceeding with RNA isolation. Alternatively, the cell pellets

were stored in 5 volumes of RNA Later (Ambion #7021) at -20oC for RNA isolation at a later

and more convenient time without compromising RNA quality or yield.

RNA ISOLATION: The following protocol was modified from BD Sciences/Clontech's AtlasTM

Pure Total RNA Labeling Kit (User Manual PT3231-1, published 24 April, 2001) for the RNA

isolation of cultured cells and subsequent DNase treatment of isolated RNA in microcentrifuge

tubes. The procedure was optimized for the RNA isolation of 40 x 106 human hepatoma HepG2

cells, which yields ~100 g RNA. Reagents in the protocol can be scaled up or down depending

on the number of cells.

RNA Isolation

Materials needed

1X PBS buffer

1.7-mL microcentrifuge tubes (RNase-DNase free)

Denaturing solution (kit)

Ice bath

Saturated phenol (Sigma #P-4682)

Vortex

Chloroform (Sigma #C-2432)

Tabletop centrifuge

Isopropanol (Sigma #I-9516)

50-mL conical tube (sterile)

80 percent ethanol (Aldrich #E702-3)

1000-L pipet and pipet tips (RNase-DNase free)

RNase-free water (kit)

100-L pipet and pipet tips (RNase-DNase free)

26 gauge needle (sterile)

10-mL syringe (sterile)