Half life of Fibronectin mRNA in Fibroblast on smooth surface

Value 5 Hours
Organism Human Homo sapiens
Reference Chou L, Firth JD, Uitto VJ, Brunette DM. Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts. J Cell Sci. 1995 Apr108 ( Pt 4):1563-73PubMed ID7615675
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).
Comments Fibronectin is a high-molecular weight (~440kDa) extracellular matrix glycoprotein that binds to membrane-spanning receptor proteins called integrins. In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans).The topography of the substratum to which cells attach plays a fundamental role in regulating cell behaviour.In an attempt to determine the effect of substratum surface topography on cell shape and the influence of the changes in cell shape on the major extracellular adhesive proteins, researchers investigated the mechanism of FN regulation at the molecular level in cells cultured on a grooved substratum produced by micromachining.
Entered by Uri M
ID 104327