| Method |
Cellular RNA was prepared by guanidinium thiocyanate (GT) according to Glisin et al. (1974) and Ullrich et al. (1977), modified as described below. Cultures in triplicate at different time points on either smooth or grooved surfaces were washed 3 times in cold PBS before the cells were lysed by 3 ml of guanidinium thiocyanate buffer (GT buffer, containing 4 M guanidinium thiocyanate, 0.025 M sodium citrate, pH 7.0, 0.5% (w/v) sarcosyl, 70 mM b-mercaptoethanol, and 5 mM vanadyl-ribonucleoside complex (VRC, BRL Inc.) as a RNase inhibitor. Cell lysates were immediately transferred into a polypropylene centrifuge tube. After brief vigorous vortexing, 0.3 ml 2 M sodium acetate, pH 4.1, 3 ml of RNase-free water-saturated phenol and 0.6 ml of chloroform-isoamylalcohol (49:1, v/v) were added separately with vortexing between each addition. After incubation on ice for 15 minutes, RNA was collected from the upper aqueous phase after centrifugation at 12,000 g for 20 minutes at 4°C, and the aqueous phase was precipitated overnight at -20°C in 1 volume of ice-cold isopropanol. The precipitated RNA pellet was rinsed with 80% cold ethanol, vacum dried, and dissolved in 100 ml RNase-free water. The total RNA yields were determined for each sample by spectroscopic analysis of 1/10th of the final sample volume. To estimate the halflife of fibronectin mRNA, cells were cultured in triplicate as described above on either grooved or smooth titanium surfaces, and after 40 hour incubation (~90% confluent) 60 mM 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (DRB, Sigma), a specific RNA polymerase II inhibitor, was added to the cultures (Overall et al., 1991). |