Comparison of snRNA-U6 and microRNA-16 for Identification of Suitable Endogenous Control Gene for microRNA Gene Expression Analysis under Dendrosomal Curcumin Treatment in Hepatocellular Carcinoma Cell Lines

Authors
M. Sadeghizadeh
M. Zamani
M. Behmanesh
M. Sadeghizadeh
M. Behmanesh
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Objective: MicroRNAs (miRNAs) are single-stranded small RNAs 18-25 nucleotides in length that regulate gene expression through translational inhibition and mRNA cleavage. Aberrant expression of miRNAs contribute to several diseases. This has increased interest in profiling the expressions of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive, quantitative technique for gene expression assessment. To correct for systematic variables such as the amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalized to an endogenous control gene which is stably-expressed across the test sample set. To avoid occurring further error in the quantification of gene expression data, it is necessary that candidate endogenous controls be validated in the samples of interest. In this study the expression of miRNA-16 and small nuclear RNA (snRNA)-U6 in hepatocellular carcinoma (HCC) cell lines under dendrosomal curcumin treatment were evaluated to identify appropriate endogenous controls for dendrosomal curcumin-related miRNA expression assays.
Methods: HCC cell lines were treated with dendrosomal curcumin. Dendrosomal curcumin entry into HepG2 and HuH-7 cells was assessed by fluorescent microscopy images. RNA was extracted and cDNA, after polyA polymerization, was synthesised. Then, we performed gene expression assays using RQ-PCR.
Results: In this treatment condition, miRNA-16 for HepG2, snRNA-U6 and the combined miRNA-16 and snRNA-U6 for HuH-7 were suitable endogenous controls.
Conclusion: These genes are appropriate endogenous controls for miRNA expression assays in HCC cell lines under treatment with dendrosomal curcumin. There are stable, non-significant expression changes of these genes.

Keywords

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Pathobiology Reserach
Volume 18, Issue 1
April 2015
Pages 67-82