RNA extraction

TRIzol Reagent was used for RNA extraction from serum-cell supernatants, serum, urine and tissues according to Ambion, Life Technologies protocol. For tissues, approximately 50 mg of tissue was homogenized with one mL of TRIzol Reagent. A 5mm stainless steel bead (Qiagen, Valencia, CA) was used with a TissueLyser LT (Qiagen, Valencia, CA) at 50 Hz for 5 minutes. One ml of TRIzol was added to urine to 5 to 15 μl of urine. One ml of TRIzol was added to 160 μl of serum from AJ-z2, AJ-z3, and AJ-z4. Two-hundred microliters of serum-cell supernatants were added to one ml of TRIzol. Samples were then incubated at room temperature for 5 minutes. Chloroform (Thermo Fisher Scientific, Waltham, MA) was added, samples were mixed, incubated for 3 minutes at room temperature and centrifuged at 12,000 x g for 15 minutes at 4°C. The aqueous phase was removed, 4 μg of glycogen (Thermo Fisher Scientific, Waltham, MA) and 100% molecular grade isopropanol added (Thermo Fisher Scientific, Waltham, MA). Samples were incubated at room temperature for 10 minutes and then centrifuged at 12,000 x g for 10 minutes at 4°C. Supernatant was removed and 75% molecular grade ethanol (Thermo Fisher Scientific, Waltham, MA) was added to RNA pellet. Samples were vortexed and centrifuged at 7500 x g for 5 minutes at 4°C. Wash was removed and air-dried. RNA was resuspended in RNase-free water and stored at -80°C for future use.