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MicroRNAs (total RNA) in cultured cancer cells were extracted using a RNA extraction kit from Qigen according to the recommended procedure, and miRNAs in the total RNA were enriched by using a cleanup kit (Qigen). Circulating miRNAs in serum were extracted using the miRNeasy RNA isolation kit from Qiagen. Briefly, 5 mL of Qiazol solution was added to 0.50 mL of serum. The mixture was thoroughly mixed and incubated for 10 min at room temperature. After 1.0 mL of chloroform was added, the mixture was vortexed for 60 s and centrifuged at 14 000 g for 15 min at 4 ¡ãC. Isolation and purification of miRNAs in the aqueous phase was achieved following the recommended procedure. Finally, miRNAs in the extracted total RNA samples were analyzed as described above. Reference values of miRNAs were obtained by qPCR (the gold standard). Direct profiling of circulating miRNAs in serum was conducted after spiking aliquots of concentrated TMD buffer to serum samples to have a final TMD buffer composition of 50 mM Tris-HCl, 5.0 mM MgCl2, and 1.0 mM DTT.

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morphological observation are also  implemented£¬ÕÒר¼Ò°É

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