Mirna Transfection Procedure

Doc. S1

Cell culture

Briefly, 5 mL of cord blood was laid over 1.077 g/mL Ficoll-Hypaque (Pharmacia Biotech, NJ, U.S.A.) gradient and centrifuged at 400 g for 25 min. Mononuclear cells were then separated and cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO-BRL, Grand Island, NY, U.S.A.) supplemented with 30% fetal bovine serum (FBS, Gibco), dexamethasone (100 nM; Sigma), L-glutamine (2 mM; Gibco), penicillin (100 U/mL; Gibco), and streptomycin (0.1 mg/mL; Gibco). The colonies of USSCs were appeared after almost 2 weeks. When cells reached 80% confluency, they were detached with 0.25% Trypsin- EDTA (Gibco) and replated 1:3.Cells were diluted to 1 million cells per vial and frozen in FBS (Gibco) plus 10% dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO, USA) at a controlled rate to 70C by using a cell freezer box. Cell stocks were transferred to liquid nitrogen storage after 24 hours.

miRNA Transfection Procedure

1. We spotted 0.5 μl miRNA mimic or inhibitor in 2 μl of RNase-free water into a single well of a 96-well plate (this will give a final miRNA mimic concentration of 5 nM or a final miRNA inhibitor concentration of 50 nM after addition of cells to complexes in step 4).

2. Then we added 0.75 μl of Transfection Reagent to 24.25 μl of culture medium without serum. In continuation, we included the diluted Transfection Reagent to the prespotted miRNA mimic/inhibitor.

3. Incubation for 5–10 min at room temperature (15–25°C) to allow formation of transfection complexes was done.

4. After that 2*10 4 cells in 175 μl of growth medium (containing serum and antibiotics) were seeded into the well, on top of the miRNA mimic/inhibitor–Reagent transfection complexes and incubated under mild shaking for 6 hours. Finally we changed the medium by fresh growth medium till 72 hours.

PCR details:

Reaction mixtures for PCR included 2.5 μl cDNA, 1x PCR buffer (AMS TM, cinnagen, Iran), 200 μM dNTPs, 0.5 μM of each of forward and reverse primers and 1U Taq DNA polymerase (Fermentas, MD, USA).Amplified DNA fragments were electrophoresed on 2% agarose gel. The gels were stained with ethidium bromide (10 μg/ml) and photographed on a UV trans-illuminator (uvidoc, UK).

Primers used for RT-PCR and real-time PCR.