Phrg tk renilla luciferase internal control plasmid

The NOTCH1 3′-UTR containing target sequences complementary to the miR-34c seed sequence was cloned downstream of the Firefly luciferase gene in the pMIR-REPORT luciferase vector (Life Technologies). Mutated NOTCH1 3′-UTR sequences were also cloned in the same vector. The indicated reporter constructs and 100-nM miR-34c mimics were cotransfected with the phRG-TK Renilla luciferase internal control plasmid (Promega Corporation) into 293T cells. Cell extracts were prepared 24 hours after transfection, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation), and the ratio of Firefly/Renilla values was reported. The Dual-Luciferase Reporter Assay was performed as described by Xu et al.^{8 (link)}

The TIMP2 3′-untranslated region (3′-UTR), containing target sequences complementary to the miR-616 seed sequence, was cloned downstream of the firefly luciferase gene in the psiCHECK-2™ luciferase vector (Promega Corporation). Mutated TIMP2 3′-UTR sequences were cloned into the same luciferase vector (TIMP2-mut). The indicated reporter constructs and miR-616 mimic or inhibitor were co-transfected with the phRGTK Renilla luciferase internal control plasmid (Promega Corporation) into 293 cell line using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The luciferase activity was analyzed following 24 h transfection using a dual-luciferase reporter assay system (Promega Corporation), according to the manufacturer's protocols.