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Objective: To identify a more consistent and homogeneous tumorigenic side population (SP) from hepatocellular carcinoma (HCC) cell line PLC/PRF-5 and screen for SP-specific microRNAs (miRNAs).Methods: Four factors in the staining process, the incubation time, shaking interval, culture time, and Hoechst 33342 concentration, were explored respectively to define the SP subtype.Cancer stem cells(CSC) characteristics of SP cells were verified by CSC marker expression and NOD/SCID mice tumorigenic ability assay.QPCR assay for 370 miRNAs was performed to identify the differential miRNA expression between SP and NSP cells.Results: All 4 factors tested influence SP percentage; 0.40% SP cells had 1.95% ABCG2, 15.92% EpCAM positive cells and 0.99% double positive cells, much higher than those in cells with 33.67% SP.While l×102 SP cells generated a tumor, at least l×103 NSP cells were needed to achieve the same.Under the same inoculation dosage, the size of tumors generated by SP cells was greater than that by NSP cells.QPCR miRNA assay identified 27 differentially expressed miRNAs.Conclusions: The present study has identified a stringent SP subtype with CSC ability from PLC/PRF/5 HCC cell line by optimizing the 4 factors of staining process.Altogether, 27 SP-specific miRNAs were determined, which might shed light on the carcinogenesis of HCC.