Facscalibur with cellquest pro 4

Short hairpin RNAs (shRNAs) were designed against the target sequences of the NANOG and HOXA9 genes. The shRNAs were controlled for sequence specificity using a BLAST search and did not show any homology to other known human genes. Plasmids expressing gene-specific shRNA were constructed using synthetic oligonucleotides cloned into the BglII/HindIII cloning sites of the pSUPER-EGFP vector (pSUPER RNAi System; OligoEngine, Seattle, WA, USA). In all RNA interference experiments, a negative control vector containing scrambled shRNA was included (pLKO.1-TRC1 and pLKO-TRC2, Supplementary Fig. S4, S5). The target sequences of the short hairpin RNAs (shRNAs) against NANOG and HOXA9 genes were as the following: shNANOG: CCG GGC TGC TAA GGA CAA CAT TGA TCT CGA GAT CAA TGT TGT CCT TAG CAG CTT TTT; shHOXA9: CCG GTG CTG ATT GTA ACG GAG TTA ACT CGA GTT AAC TCC GTT ACA ATC AGC ATT TTTG. The shRNA expression constructs were transfected using an electroporation machine (Microporator; Digital Bio Technology, Suwon, Korea) with 6–8 μg of DNA at 1100–1300 V for 20–30 mS¹⁸ . Transfection efficiency was determined by flow cytometric analysis on fluorescent cells with a flow cytometer (FACSCalibur with CellQuest Pro 4.0.2; Becton Dickinson, Franklin Lakes, NJ, USA). The inhibition of gene expression was evaluated using immunoblotting.

Flow cytometric analysis was performed (Becton Dickinson, Mountain View, CA, USA) as described previously^{18 ,20 (link)}. Cell viability was determined by the trypan blue exclusion test. Apoptosis and other forms of cell death were evaluated by measuring the DNA content using annexin V and propidium iodide (PI) affinity as previously described^{21 (link)}. Briefly, each sample of 2.4 × 10^{6 (link)} cells was transfected with candidate gene-specific shRNA or control vector, and then cultured in 6 ml of medium. Each sample of 1.5 ml was collected after 48–72 h. The sample was then centrifuged, and the pellet was incubated with staining solution (PI [50 μg/ml]; 0.1% sodium citrate; 0.1% triton) overnight at 4 °C in the dark. Core DNA content was measured using a logarithmic amplification in the FL2 (for annexin V) and FL3 (for PI) channels of the flow cytometer (FACSCalibur with CellQuest Pro 4.0.2; Becton Dickinson)^{18 ,20 (link)}. Cell-cycle analysis was also measured using flow cytometry. The distribution of the DNA content of individual cells was stained with PI and measured by using a linear amplification in the FL3 channel.