Dharmafect duo transfection reagent

HEK293T cells were seeded in 96-well plates at 20,000 cells per well. The following day, crRNAs and tracrRNA (GE Healthcare Dharmacon, #U-002000-50) were resuspended with 10 mM Tris-HCl pH 7.4 buffer (GE Healthcare Dharmacon, #B-006000-100) to 10 μM. For transfection in triplicate wells of duplicate cell plates, a deep-well plate was prepared with a 1:1 mixture of tracrRNA and each crRNA (5 μM each) diluted in MEM-RS medium (GE Healthcare HyClone, #SH30564.01) to 250 nM along with 1.4 μg of plasmid (70 μL total volume per well). DharmaFECT Duo transfection reagent (GE Healthcare Dharmacon, #T-2010-01) was diluted with MEM-RS medium (240 μL of DharmaFECT Duo transfection reagent was added to 3.76 mL of MEM-RS medium). Diluted transfection reagent (70 μL) was added to the crRNA:tracrRNA in the deep-well plates and incubated at room temperature for 20 minutes. After incubation, 560 μL of full growth medium was added per deepwell to obtain a complete transfection mixture (700 μL total) for triplicate transfection, in 2 plates. In each well, the medium on the plated cells was replaced with 100 μL of transfection mixture (final concentration: 0.6 μL of the DharmaFECT Duo transfection reagent, 200 ng of plasmid, and 25 nM of the crRNA:tracrRNA in each well). The cells were incubated at 37° C with 5% CO₂ for 72 hours.

For luciferase reporter assays, MCF-7 cells were co-transfected with HDAC4 3′UTR luciferase vector (GeneCopoeia, Catalog # HmiT023167-MT05) and pre-miR-10b or miRNA negative control, using DharmaFECT Duo Transfection Reagent (Dharmacon). The vector has HDAC4 3′ UTR sequence inserted downstream of the secreted Gaussia luciferase (GLuc) reporter gene system, driven by SV40 promoter for expression in mammalian cells. A secreted Alkaline Phosphatase (SEAP) reporter, driven by a CMV promoter, is also cloned into the same vector (pEZX-MT05) and serves as the internal control. 48 h post- transfection, Gluc and SEAP luciferase activities were assayed using Secrete-Pair^™Dual Luminescence Assay Kit (GeneCopoeia), following exactly the same procedure as described in the vendor’s protocol.