Factors influencing circulating MicroRNA level in the studies of hepatocellular carcinoma biomarker.

Factors influencing circulating miRNA as a diagnostic marker of hepatocellular carcinoma (HCC) are still unknown. We used two kits to extract total RNA of 90 paired serum and plasma samples of volunteers in different statuses including healthy, hepatitis, cirrhosis and HCC. U6, miR-183 and cel-miR-39 expression levels were detected by qRT-PCR. Calculate was the relative expression level of miR-183 and recovery of cel-miR-39 with 2-∆∆CT method and standard curve method, respectively.MiRNeasy Serum/Plasma Kit was superior to miRcute miRNA Isolation Kit in the quality of the total RNA and the expression levels of U6 and miR-183, with recovery of external reference cel-miR-39 of the former in serum and plasma was the latter's 3.95-fold and 3.15-fold, respectively. The recovery of cel-miR-39 was significantly higher in serum than that in plasma in all groups except the HCC. But the trend of U6 expression of four groups in serum was different from that in plasma. There were significant differences in absorbance in 260nm, the ratio of absorbance in 260nm and 280nm (260/280), the ratio of absorbance 260nm and 230nm (260/230), the ct value of ​​U6 and the recovery of cel-miR-39 among groups in serum or plasma. Sensitivity and specificity of miR-183 as a diagnostic marker of HCC were 57.9% and 76.2% in serum, and 78.9% and 81.0% in plasma, respectively.Caution should be taken when comparing miRNA data generated from different extraction kits, sample types or disease statuses in the study of miRNA as a diagnostic marker of HCC.