Dual luciferase reporter assay system psicheck 2 vector

The dual luciferase assay was performed using the dual-luciferase reporter assay system (psiCHECK-2 vector, Promega). A 850-bp fragment of the MTHFR 3' UTR was amplified from genomic DNA using the following primer sequences: forward, 5 ‫׳‬ -GGACTAGTGGTTGTTGCCAACTAAGCCC-3 ‫׳‬ ; reverse, 5 ‫׳‬ -TTCAAGCTTTCCAGGGAGTGATGACAGAG-3 ‫׳‬ . The PCR product was inserted into the psiCHECK-2 vector downstream of the luciferase gene sequence. All of the constructs were verified by DNA sequencing. According to the MTHFR genotypes, constructed vectors were termed psiCHECK-AA and psiCHECK-GG, respectively. HEK-293 cells were plated in 96-well clusters. Lipofectamine 2000 was used to transfect the cells with 80 ng of psiCHECK-AA or psiCHECK-GG reporter constructs,(Invitrogen, USA)hsa-miR-214 or controls (all at 30 nM, Ambion, USA), and 80 ng of a psiCHECK-2 luciferase vector. After 48 hoftransfection, the cells were washed twice and lysed with passive lysis buffer. Firefly luciferase activity was determined using the dual-luciferase reporter assay system and a luminometer (Promega), as we previously described [27] . The relative luciferase activity was calculated by normalizing the firefly luciferase activity against the internal control luciferase activity.

Luciferase reporter assays were carried out using the Dual-Luciferase Reporter Assay System (psiCHECK-2 vector; Promega, Madison, WI, USA). A fragment of the Mef2c 3′UTR containing the predicted binding site for miR-27a and the binding site mut 3′UTR were inserted into psiCHECK2 vector. All constructs were verified by DNA sequencing. The psiCHECK-2 vector containing wild-type (wt) or mut Mef2c was transfected into cells with or without the synthetic miR-27a mimic. Thirty-six hours after transfection, luciferase activity was detected using a dual-luciferase reporter assay system and normalized to Renilla activity.