PCR screening of miRNA in AIONFH cases and health control
MiRCURY LNATM Universal RT microRNA PCR Panels is utlized to analyze the spectra of miRNAs in serum samples from AIONFH patients and applied the random variance model (RVM) t-test to filter the differentially expressed miRNAs. The distinguishable miRNA expression profiles among AIONFH patients and healthy controls were demonstrated by the results of hierarchical clustering. Initial analysis revealed 10 miRNAs that were differentially expressed. Seven miRNAs (miR-127-3p, miR-1, miR-654-3p, miR-628-3p, miR-432-5p, miR-582-3p, and miR-323a-3p) were found to be significantly downregulated (P<0.01), while three miRNAs (miR-483-5p, miR-483-3p, and miR-885-5p) were markedly upregulated (P<0.01)
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Total miRNAs were measured from two pooled serum groups each consisting of a pool of 5 AIONFH serum samples and a pool of 5 healthy serum samples to identify differentially expressed miRNAs. miRNAs were isolated and extracted from serum in a standard method according to the manufacturers’ recommendations (Invitrogen, Carlsbad, CA, USA). Briefly, the serum samples from both groups were centrifuged at 13000×g for 5 min at 4°C to eliminate pellet and cell debris. The supernatant serum was collected and added to TRIzol-LS (Invitrogen, Carlsbad, CA, USA), and then high-speed centrifugation was performed to obtain the aqueous phase. To precipitate the total RNA, serum was mixed with isopropyl alcohol and subjected to one further high-speed centrifugation at 10000×g for 5 min at 4°C. The total RNA was eluted in RNase-free water and stored at -80°C until analysis. The amount and purity of RNA was estimated by ultraviolet spectrophotometer. MiRNAs were profiled with miRCURY LNATM Universal RT microRNA PCR Panels (Exiqon, Vedbaek, Denmark). RNA samples were diluted to 1.5-1.8 ng/μl using nuclease-free water. The specific miRNA primers for reverse transcription (RT) were used to amplify miRNAs in an RT reaction mix (Exiqon, Denmark) and subjected to amplification using SYBR™ Green Master Mix (Exiqon, Denmark) with an ABI PRISM 7900 Real-time PCR System (Applied Biosystems, USA). Quantitative miRNA expression levels were analyzed by 2^-ΔCt/ΔCt calculation with GenEx qPCR analysis software (Exiqon, Vedbaek, Denmark). Fold changes>2-fold or P<0.05 were used to identify miRNAs that had significantly altered expression between the patient group and the healthy control group.