Genetic variants, including mirSNPs have been functionally tested using a reporter plasmid such as the pIS-0 vector (Yekta et al., 2004; Adams et al., 2007; Ramamoorthy et al., 2012). This plasmid contains the firefly luciferase gene whose expression can be quantified either by qPCR or by the luciferase reporter assay. The reference or variant allele version of the predicted miRNA binding sites were cloned into the 3′ UTR of the luciferase gene within the plasmid. The plasmids were then transfected into cells as described above. Forty-eight hours after transfection, cells were lysed in situ and Dual-Luciferase® assays were performed per manufacturer’s instructions (Promega, Madison, WI, United States). The luciferase reporter activity was measured using a 96-well plate-reader (BioTek, Winooski, VT, United States). The firefly luciferase activity was normalized to that of Renilla luciferase in each well. The ratio of the normalized luciferase activity from the variant and reference plasmid provides a relative measure of SNP-mediated differential mRNA expression.
