Abstract
BackgroundMicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2.ResultsA panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p.ConclusionWe identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-1848-2) contains supplementary material, which is available to authorized users.
Highlights
MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics
We investigated the overall expression at two different time points (48 and 72 h) in the control HepG2 tet-on cell line and the Hepatitis B virus (HBV)-replicating cell line (DOXY) (See Additional file 1 Figure S1 for heatmap, and Additional file 2 Table S1 for raw CT values)
We evaluated the overall stability of each microRNA by calculating the standard deviation (SD) for all samples
Summary
MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. We previously identified a panel of differentially expressed microRNAs in plasma from children with CHB [14] and showed that a number of Jacobsen et al BMC Res Notes (2016) 9:38 the identified microRNAs had liver-specific target genes [15]. Instead we use an in vitro HBV-replicating liver cell model to investigate the biological role of microRNAs with a possible influence on the pathogenesis of CHB in children
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