Pmir report luciferase pmir r l control vector

Total cDNA of H9C2 cells was used to amplify TGFβ1 gene by PCR, which was then inserted into the pMIR-REPORT™ Luciferase (pMIR-R-L) control vector (Ambion). The Renilla luciferase vector was employed for normalization. The cells in 24-well plates were transfected with 0.2 μg of pMIR-R-L vector and 0.04 μg of control vector in the presence of Lipofectamine 2000. Moreover, pCMV6-ALK4 or inhibitor-ALK4 was used in each well. At 48 h after transfection, the dual-luciferase reporter assay system (Promega) was used to measure the pMIR-R-L and Renilla luciferase activities consecutively.

3′UTR segments of the Wnt gene were amplified by PCR from the total cDNA of BMSCs and inserted into the pMIR-REPORT™ Luciferase (pMIR-R-L) control vector (Ambion) using HindIII and SpeI as restriction sites. Target segments and mutant inserts were confirmed by sequencing. Renilla luciferase vector was used for normalization. The cells were co-transfected in 24-well plates using Lipofectamine 2000 according to the protocol of the manufacturer with 0.2 µg pMIR-R-L vector and 0.04 µg control vector. pMIR-R-L and Renilla luciferase activities were measured consecutively using the dual-luciferase reporter assay system (Promega, MA, U.S.A.) 48 h after transfection.
Dual luciferase assay was used to detect the signal of Notch pathway using a commercial Notch Pathway Reporter kit (BPS Bioscience Corp., San Diego, California), according to specification.