MiRNA (Bone) validation of AIONFH cases by Real-time PCR
We further performed qRT-PCR in necrotic bone tissue samples to confirm the expression of potential miRNAs. Our results showed that miR-127, miR-628-3p, and miR-1 were downregulated (P<0.05), and miR-885-5p, miR-483-3p, and miR-483-5p were upregulated (P<0.05). The expression of these miRNAs maintained the consistent tendency in bone as in serum. However, no significant difference in miR-432-5p expression was observed in necrotic bone tissue samples compared with that in serum. Hence, our results revealed a close relationship between miRNAs and AIONFH. Thus, we focused on these six miRNAs for further analysis in the following experiments.
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To further validate the miRNA array data, the differentially expressed miRNAs were analyzed with quantitative RT-PCR (qRT-PCR) in duplicate using an miRNA assay kit (GenePharma, Shanghai, China) in 10 bone samples (5 AIONFH patients and 5 healthy controls with femoral neck fracture). Serum and femoral head isolated or detached from patients and volunteers were stored in -80°C. Necrotic bone tissue was partially evaluated by histological examination and tissue with empty lacuna rate more than 50% was identified as necrotic area. To isolate miRNA from serum and bone tissue, extraction with TriReagent (Invitrogen life technologies) was performed after milling of the tissue using liquid nitrogen. The RT reaction was performed under the following reaction conditions: 30 min at 25°C, 30 min at 42°C, and 5 min at 85°C, followed by maintenance at 4°C. The selected miRNAs were confirmed with SYBR Green I dye (Takara, Dalian, China) with an ABI PRISM 7300 Real-time PCR System (Applied Biosystems, USA) at 95°C for 3 min, followed by 40 cycles at 95°C for 12 s and 62°C for 40 s. Cel-miRNA-39-3p, a nonhuman miRNA, was spiked into the RNA samples as a control for the extraction and amplification steps. GAPDH was used for normalization of serum samples following the miRNA PCR array analysis.