One hundred microliters of 10% tissue homogenate were mixed with 900 ml of TRIzol reagent (Invitrogen, Carlsbad, CA). After addition of 200 ml of chloroform, tubes were shaken vigorously by hand and centrifuged at 15,000 rpm for 10 min at 4uC. Following centrifugation, the aqueous phase was transferred to a new tube and 500 ml of isopropanol was added to the tubes. Samples were centrifuged at 15,000 rpm for 25 min at 4uC. RNA pellets were washed with 75% ethanol and dried. Thirty microliters of RNasefree water was added to dissolve the RNA pellet. The samples were then treated with RQ1 RNase-Free DNase (Promega, Madison, WI), and the RNAs were purified by addition with phenol-chloroform.