siRNA / miRNA gene silencing Human - hES cell line H1 (WA01) LIN28

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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ON-TARGETplus Human LIN28A (79727) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	The hESC lines H1 listed in the NIH hESC registry under the names of WA01 respectively) were obtained from WiCell. The iPSC line was generated at the Yale Stem Cell Center using Yamanaka factors (Takahashi et al. 2007). The cells were cultured in an undifferentiated state in Matrigel (BD Pharmingen CA)-coated plates under feeder-free and defined component conditions (Yao et al. 2006) at 37°C in hESC culture incubators (5% CO2, 5% O2, and 90%–95% humidity). Cell growth media were composed of Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen), supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin (P/S), 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1×), and B27 supplements (1×) (Invitrogen). Cells were maintained with daily medium change and passaged weekly by dissociation into small clumps using 1 mg/mL dispase (StemCell Technology). The hESCs and iPSCs used were between passages 30 and 60 and 10 and 15, respectively. Free of mycoplasma contamination, the cells exhibited normal karyotype, expressed common ES markers, and were able to differentiate into the three germ layers (data not shown). siLin28 (ON-TARGETplus SMARTpool) and siCon were purchased from Dharmacon. To prepare siRNA/lipid solutions for each well of a six-well plate, 9 or 36 pmol of siRNAs were diluted in 50 μL of OPTI-MEMI (Invitrogen) and incubated at room temperature for 5 min. In a separate tube, 5 μL of Lipofectamine 2000 (Invitrogen) was diluted in 50 μL of OPTI-MEMI and incubation carried out for 5 min at room temperature. The contents of the two tubes were combined by gentle pipetting and incubated at room temperature for 30–50 min. The resulting 100 μL of transfection solution was used to resuspend the cell pellet (1 × 106) produced after Accutase dissociation (see above). For siRNA transfection using the standard method, cells were seeded in a 6-well plate 5 d prior to transfection. On the day of transfection, 100 μL of siRNA/lipid solution diluted in 1.5 mL of growth medium was added to each well of cells grown as single-layer colonies attached to the plate. ROCK inhibitors were not involved before, during, or after the transfection. RNAs were extracted for analysis 72 h after the transfection.

Protocol tips
The hESC lines H1 listed in the NIH hESC registry under the names of WA01 respectively) were obtained from WiCell. The iPSC line was generated at the Yale Stem Cell Center using Yamanaka factors (Takahashi et al. 2007). The cells were cultured in an undifferentiated state in Matrigel (BD Pharmingen CA)-coated plates under feeder-free and defined component conditions (Yao et al. 2006) at 37°C in hESC culture incubators (5% CO2, 5% O2, and 90%–95% humidity). Cell growth media were composed of Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen), supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin (P/S), 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1×), and B27 supplements (1×) (Invitrogen). Cells were maintained with daily medium change and passaged weekly by dissociation into small clumps using 1 mg/mL dispase (StemCell Technology). The hESCs and iPSCs used were between passages 30 and 60 and 10 and 15, respectively. Free of mycoplasma contamination, the cells exhibited normal karyotype, expressed common ES markers, and were able to differentiate into the three germ layers (data not shown). siLin28 (ON-TARGETplus SMARTpool) and siCon were purchased from Dharmacon. To prepare siRNA/lipid solutions for each well of a six-well plate, 9 or 36 pmol of siRNAs were diluted in 50 μL of OPTI-MEMI (Invitrogen) and incubated at room temperature for 5 min. In a separate tube, 5 μL of Lipofectamine 2000 (Invitrogen) was diluted in 50 μL of OPTI-MEMI and incubation carried out for 5 min at room temperature. The contents of the two tubes were combined by gentle pipetting and incubated at room temperature for 30–50 min. The resulting 100 μL of transfection solution was used to resuspend the cell pellet (1 × 106) produced after Accutase dissociation (see above). For siRNA transfection using the standard method, cells were seeded in a 6-well plate 5 d prior to transfection. On the day of transfection, 100 μL of siRNA/lipid solution diluted in 1.5 mL of growth medium was added to each well of cells grown as single-layer colonies attached to the plate. ROCK inhibitors were not involved before, during, or after the transfection. RNAs were extracted for analysis 72 h after the transfection.

Protocol tips

The hESC lines H1 listed in the NIH hESC registry under the names of WA01 respectively) were obtained from WiCell. The iPSC line was generated at the Yale Stem Cell Center using Yamanaka factors (Takahashi et al. 2007). The cells were cultured in an undifferentiated state in Matrigel (BD Pharmingen CA)-coated plates under feeder-free and defined component conditions (Yao et al. 2006) at 37°C in hESC culture incubators (5% CO2, 5% O2, and 90%–95% humidity). Cell growth media were composed of Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen), supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin (P/S), 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1×), and B27 supplements (1×) (Invitrogen). Cells were maintained with daily medium change and passaged weekly by dissociation into small clumps using 1 mg/mL dispase (StemCell Technology). The hESCs and iPSCs used were between passages 30 and 60 and 10 and 15, respectively. Free of mycoplasma contamination, the cells exhibited normal karyotype, expressed common ES markers, and were able to differentiate into the three germ layers (data not shown). siLin28 (ON-TARGETplus SMARTpool) and siCon were purchased from Dharmacon. To prepare siRNA/lipid solutions for each well of a six-well plate, 9 or 36 pmol of siRNAs were diluted in 50 μL of OPTI-MEMI (Invitrogen) and incubated at room temperature for 5 min. In a separate tube, 5 μL of Lipofectamine 2000 (Invitrogen) was diluted in 50 μL of OPTI-MEMI and incubation carried out for 5 min at room temperature. The contents of the two tubes were combined by gentle pipetting and incubated at room temperature for 30–50 min. The resulting 100 μL of transfection solution was used to resuspend the cell pellet (1 × 106) produced after Accutase dissociation (see above). For siRNA transfection using the standard method, cells were seeded in a 6-well plate 5 d prior to transfection. On the day of transfection, 100 μL of siRNA/lipid solution diluted in 1.5 mL of growth medium was added to each well of cells grown as single-layer colonies attached to the plate. ROCK inhibitors were not involved before, during, or after the transfection. RNAs were extracted for analysis 72 h after the transfection.

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