Validation of miRNA Expression in Vascular Calcification

We then confirmed miRNA expression of selected miRNA that controlled multiple genes and/or controlled pathways which are known to be important in vascular calcification, by real time PCR as previously described [17 (link)] using TaqMan miRNA assays (Applied Biosystems, Foster City, CA). Forty ng of total RNA were used for reverse transcription to synthesize complementary DNA using TaqMan miRNA-specific primers and the Taq reverse transcription kit (Applied Biosystems, Foster City, CA). Target-specific PCR primers (miR-667, miR-702, miR-3562, miR-3568 and miR-3584) were obtained from Applied Biosystems. Real-time PCR amplifications were performed using TaqMan miRNA assays (TaqMan MGP probes, FAM dye-labeled) using Applied Biosystems 7500 Real-Time PCR systems (Applied Biosystems). The cycle number at which the amplification plot crosses the threshold was calculated (C_T), and the ΔΔC_T method was used to analyze the relative changes in gene expression and normalized by U6, a non-human ubiquitous miRNA.