Fracture Callus miRNA Expression

Based on the array results, the top four miRNAs on days 5 and 11 were selected for further real-time PCR analysis. The newly generated calluses collected on post-fracture days 5, 7, 11, 14, 21, and 28 were harvested (n = 6 per group at each time point). The calluses were homogenized, and total RNA was extracted. RNA was reverse-transcribed to single-stranded cDNA using the miRCURY LNA Universal RT microRNA PCR Kit (Exiqon, Vedbaek, Denmark). Real-time PCR analysis was performed in duplicate with a StepOne Sequence Detector (Applied Biosystems, Branchburg, NJ, USA), using SYBR Green Master Mix and microRNA LNA PCR primer sets (both from Exiqon). As an internal control gene in miRNA PCR assays, U6 was used [13 (link), 14 (link)]. The conditions of the PCR amplification were 95 °C for 30 sec, followed by 40 cycles of 95 °C for 30 sec and 75 °C for 15 sec. Changes in miRNA expression levels were calculated using the comparative ΔΔCT method [13 (link), 14 (link)] and is presented as the fold change relative to the level of the corresponding miRNA in the control group on post-fracture day 5.