miRNA profile of rat vascular smooth muscle cell stimulated by 2.6 mM high-phosphate

We compared time-series miRNA expression profile in high-phosphate–stimulated rat vascular smooth muscle cells at 0, 3, and 12 hr respectively. Total RNA of rat VSMCs stimulated with 2.6 mM inorganic phosphate for 0, 3 or 12 hr was harvested by the Trizol method (Invitrogen) or use of the miRNeasy mini kit (QIAGEN). The samples were labeled by use of the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.16.0) (Exiqon, Denmark). After washing slides were scanned by use of the Axon GenePix 4000B microarray scanner. Expressed data were normalized by the Median normalization method, and then relative fold change at each time was detected by level of fluorescence in the phosphate-treated group normalized to that in the control group. Finally, hierarchical clustering was performed for miRNA expression profiling. Student t test was used to compare differences between 2 groups, and a 1.5-fold change was considered significant.