Ribonuclease t1

Ovaries were separated from oviducts, snap frozen on dry ice, and stored at -80°C until shipment on dry ice to the National Center for Toxicological Research (Jefferson, AR). There, ovaries were homogenized in 0.2 ml of extraction buffer, consisting of 0.5 mg/ml proteinase K, 20 mM NaCl, 1 mM CaCl₂, and 10 mM Tris pH 8.0. Samples were incubated ~16 hrs at 37°C, then extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol-precipitated. Samples were resuspended in 100 Nl of RNase buffer: 10 mg/ml RNase A (Sigma, St. Louis, MO), 600 units/ml Ribonuclease T1 (Sigma), 100 mM sodium acetate, and 50 mM Tris-HCl (pH 8), incubated ~16 hours at 37 °C, then re-extracted with phenol/chloroform/isoamyl alcohol as described above. Each DNA sample was ethanol precipitated, then resuspended in 50 Nl of TE buffer (5 mM Tris, 0.5 mM EDTA, pH 7.5). DNA samples were digested with HindIII according to the manufacturer’s instructions (New England Biolabs, Beverly, MA). Finally, the DNA was phenol/chloroform/isoamyl alcohol extracted as described above, ethanol-precipitated and resuspended in 40 Nl of TE buffer. DNA concentrations were measured spectrophotometrically.

DNAs from normal lung and lung tumors were isolated on separate days, employing precautions to limit potential cross-contamination. Tissues were homogenized using an Omni THQ tissue homogenizer (Omni International, GA, USA) and 6 ml of proteinase K buffer (1 mg/ml proteinase K, 100 mM NaCl, 25 mM EDTA [pH 8], and 1% SDS) per gram of tissue. Samples were incubated approximately 16 h at 37°C, and extracted with an equal volume of a 25:24:1 phenol/ chloroform/isoamyl alcohol mixture. Samples were resuspended in 200 µl of RNase buffer, comprised of 10 mg/ml RNase A (Sigma, MO, USA), 4.2 units/µl ribonuclease T1 (Sigma), 100 mM sodium acetate and 50 mM Tris-HCl (pH 8), incubated approximately 16 h at 37°C, and then extracted as described above. The DNA was ethanol precipitated and resuspended in TE buffer (5 mM Tris, 0.5 mM EDTA, pH 7.5). Finally, all genomic DNAs were restriction digested, phenol/chloroform/isoamyl alcohol extracted, ethanol-precipitated and resuspended in TE buffer at a final concentration of approximately 500 ng/µl.