Modulas microplate luminometer

The ALDOA 3′UTR fragment was cloned into the psi-CHECK2 dual-luciferase reporter system between XhoI and SpeI restriction sites using the primers found in Supplementary Table 7. A miRIDIAN microRNA mimic for miR-122-5p was obtained for the 22nt isoform or custom synthesized for the 21nt isoform (GE Healthcare). For transfection, 250 ng of psi-CHECK2 reporter plasmids were co-transfected with a final concentration of 25 nM of synthetic miRNA mimics in 24-well plates of HEK293 cells using the Tranist-TKO transfection reagent (Mirus). Cells were lysed in 1× Passive Lysis Buffer and evaluated using a Dual Luciferase Reporter Assay (Promega) 24 hours after transfection, detected on a Modulas Microplate Luminometer (Turner Biosystems).

Dual-luciferase assays (Promega) were performed 24 hours after transfection according to manufacturer′s protocol and detected by a Modulas Microplate Luminometer (Turner Biosystems). For transfection in a 24-well plate, 250 ng of psiCHECK reporter plasmids were co-transfected with 250 ng of miRNA overexpression plasmids (Sh-constructs or pEZX-constructs) in E10.5 MEF cells using Trans IT-LT1 (Mirus Bio) or in HEK 293 and Huh7 cells using Lipofectamine 2000 (Life Technologies). Cell seeding was performed at a concentration of 2.5 × 10⁴ cells for MEF, 1.25 × 10⁴ cells for Ago2^−/− and 5 × 10⁴ cells for HEK 293, A549, Dicer knockout cells and Huh7 per well in a 24-well plate. For reporter assay in Dicer knockout cells, the cells were co-transfected using Lipofectamine 3000 (Life Technologies) with a E2f63′UTR reporter construct (100 ng), 20 nM duplex synthetic miR-151-5p and increasing amounts of pEZX-DM starting from 50 ng. For the miR-151-5p inhibitor study, a miRVANA miRNA inhibitor against miR-151-5p was used (MH11537 from Life Technologies).
A fully phosphorothiolated 18 bp LNA/DNA mixmer (Exiqon) (sequence provided in Supplementary Table 5) containing 7 LNA bases predicted to block the binding of miR-151-3p to the target E2f6 3′UTR site without inducing RNAse H cleavage^{46 (link)} was cotransfected in MEF cells at 50 nM using Trans IT-TKO (Mirus Bio).