Co-transfection of 3' UTR GoClones With miRNA Mimic in Adherent Cells

Select an appropriate highly transfectable adherent cell line with low endogenous expression of the target microRNA. Choose the microRNA mimic and non-targeting control pair, then select pre-cloned 3' UTR GoClone reporter constructs and control constructs from the catalog.

On day one, seed low passage adherent cells in a white 96-well tissue culture plate at 100 microliters per well so they reach 80-100% confluence 24 hours later. Seed parallel cells in a clear plate to verify confluence.

On day two, thaw GoClone reporter constructs and microRNAs at room temperature. Dilute microRNAs to 2 micromolar in RNA-free water. Prepare the transfection reagent mixture by combining serum-free media and DharmaFECT Duo, then incubate for 5 minutes at room temperature.

Create the DNA-RNA mixture for each assay by combining GoClone reporter construct with microRNA mimic to achieve desired final concentration in 10 microliters total volume. Combine the transfection reagent mixture with the DNA-RNA mixture and incubate for 20 minutes at room temperature.

Add 80 microliters of pre-warmed antibiotic-free complete media to each assay well for a total volume of 100 microliters per well, resulting in final mimic concentrations of 10-15 nanomolar.

Carefully remove media from each well of seeded cells and replace with 100 microliters of the appropriate final transfection mixture. Return the plate to the incubator.

Incubate the transfected cells for 24 to 48 hours. Add Light Switch luciferase assay reagent directly to the cells and culture, then read the luminescent signal on a plate luminometer to identify which 3' UTRs are targeted by the microRNA.