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Abstract
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The Dual-Luciferase Reporter (DLR) assay system provides an efficient means of performing dual-reporter assays as a method of studying RNA interference. It takes the advantage of no demand for antibodies of interest. In this method, the target gene of a specific miRNA is cloned in 3ʹUTR of renilla luciferase reporter. Then, the resulted construct and the miRNA of interest were co- transfected to the host cells. Targeting the gene by miRNA could be detected by decrease in renilla luciferase luminescence. The photinus luciferase gene was used as internal control reporter for normalizing other variables in this system. Our in silico studies predicted that hsa-miR-30d can target 3ʹ UTR of human calumenin transcripts. To evaluate the capability of this miRNA to knockdown calumenin in vivo, 3ʹ UTR of calumenin was amplified and cloned as 3ʹ UTR of Renilla luciferase gene. The resulted construct (psicheck-2 3´UTR calu) was transfected to Hek293T cells and the luminescence of this reporter was measured in the presence and absence of hsa-miR-30d transfection. In addition, we did the same procedure for mock and off-target vectors, and took the advantage of a control negative miRNA to normalize the assay and determine the specificity of hsa-miR-30d in targeting human calumenin transcripts. The results of assays with mock and off-target vectors demonstrated that neither hsa-miR-30d nor control negative miRNA had knockdown effect on the luminescence of renilla luciferase. Whereas, co-transfection of psicheck-2 3´UTR and hsa-miR-30d, resulted in 0.13- 0.62 luminescence compared to control cells. Also, we observed a 0.74- 1.03 luminescence in assays with control negative miRNA. Altogether, data obtained in this study demonstrated 38% - 87% knockdown of calumenin by hsa-miR-30d, and the results of control experiments confirmed the specificity of this reaction.
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