Overexpression of miR-195, miR-497 in MSCs

Human primary MSC (Lonza) (0.5×10⁶) were mixed with Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) in an electroporation cuvette and electroporated using OPTI-MEM I (Invitrogen, Life Technologies) in a Gene Pulser Xcell Electroporation Systems (Bio-Rad) with the following conditions: voltage - 250 V, capacitance - 950 μF, resistance - 200 Ω [46 (link)].
For miR-195 antagonist experiments, 100nM anti-miR-195 (mirVana^® miRNA inhibitor, ThermoFisher Scientific) or SCR Negative Control (Anti-miR Negative Control #1, ThermoFisher Scientific) were used to transfect MSC using Lipofectamine 2000 reagent (Invitrogen), according to manufacturer's instructions.
MC3T3 cells (3×10⁶) plated in a 10-cm culture dish were transfected with 50 nM Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies). Media without antibiotics was used.
Cells were collected after 12 hours and plated for the different assays. All Electroporations/Transfections were confirmed by RT-qPCR.

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Almeida M.I., Silva A.M., Vasconcelos D.M., Almeida C.R., Caires H., Pinto M.T., Calin G.A., Santos S.G, & Barbosa M.A. (2015). miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis. Oncotarget, 7(1), 7-22.

Publication 2015

OPTI-MEM I Gene Pulser Xcell Electroporation Systems mirVana® miRNA inhibitor Lipofectamine 2000 reagent Lipofectamine 2000 transfection reagent

Anti mir Antibiotics Assays Cells Dish Electroporations Gene systems Human Lipofectamine 2000 Mirna Pre mirna Transfections

Corresponding Organization : Universidade do Porto

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Transfection of Primary Human MSCs with Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497, or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR)
Transfection of MC3T3 cells with Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR)
Transfection of Primary Human MSCs with 100 nM anti-miR-195 (mirVana® miRNA inhibitor) or SCR Negative Control

dependent variables

Outcome measurements for the different assays

control variables

Cell type (Primary Human MSCs and MC3T3 cells)
Cell seeding density (0.5x10^6 Primary Human MSCs, 3x10^6 MC3T3 cells)
Electroporator settings (voltage - 250 V, capacitance - 950 μF, resistance - 200 Ω)
Transfection reagent (Lipofectamine 2000)

controls

Positive control: Pre-miR miRNA Precursor Negative Control (Scrambled - SCR)
Negative control: SCR Negative Control (Anti-miR Negative Control #1)

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