Dual RNA and DNA Extraction from Frozen Tissue

Frozen liver tissue was placed in a lysing tube containing ceramic beads (Lysing Matrix D, MP Biomedicals, Santa Ana, CA, USA) with guanidine thiocyanate and phenol (Tri Reagent, Molecular Research Center, Cincinnati, OH, USA) and pulverized in a MagNA Lyser bead beater (Roche Life Science, Penzberg, Germany) at 6500 rpm for 45 s. The supernatant was then incubated with a 1/10 volume of bromochloropropane (Molecular Research Center) for 5 min and the organic and aqueous phases were separated via centrifugation at 12,000× g for 15 min at 4 °C. The RNA-containing aqueous phase was removed, and RNA was precipitated with isopropanol. RNA was then washed twice with 75% ethanol and air-dried before resuspending in 10 mM Tris, pH 8.0. The DNA-containing mixture of interphase and organic phase was incubated with a buffer of 4 M guanidine thiocyanate, 50 mM sodium citrate, and 1 M Tris and centrifuged at 12,000× g for 15 min at 4 °C. DNA was washed twice in 75% ethanol and air dried before resuspension with 10 mM Tris, pH 8.0.