MiRNA (serum) validation of AIONFH cases by Real-time PCR
To further validate the array results, qRT-PCR was performed. Our results showed that miR-127, miR-628-3p, miR-1, and miR-432-5p were downregulated (P<0.05), miR-885-5p, miR-483-3p, and miR-483-5p were upregulated (P<0.05), and miR-654-3p, miR-323a-3p, and miR-582-3p were unchanged. Collectively, these qRT-PCR results confirmed seven differentially expressed miRNAs using microRNA PCR Panels; however, three miRNAs were not significantly different based on qRT-PCR.
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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 30 serum samples (15 AIONFH patients and 15 healthy controls with femoral neck fracture) and 20 bone samples (10 AIONFH patients and 10 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.