Total RNA was isolated from hESC-derived neurons with TRIzol reagent (Invitrogen, Shanghai, China) and reverse-transcribed into cDNA using oligo (dT)15 and ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) was used to quantify amounts of mRNA. The qRT-PCR was performed using the ABI PRISM 7900 Fast Real-Time PCR system (Applied Biosystems, Shanghai, China) and the Power SYBR Green PCR Master Mix (Applied Biosystems, Shanghai, China) according to the manufacturer’s instructions. Mature miR-665 was detected by stem-loop qRT-PCR analysis using the Taqman Human MicroRNA Assay kits (Applied Biosystems, Shanghai, China). Small nucleolar RNU48 served as an endogenous reference RNA for normalizing the cellular content of other miRNAs.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling (TUNEL)
hESC-derived neurons were fixed in 4% paraformaldehyde for 1 h at room temperature and then permeabilized in 0.2% Triton X-100/phosphate-buffered solution for 15 min. TUNEL staining was performed according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, POD; Roche). Stained cells were analyzed using the BD FACSAria Cell Sorter.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling (TUNEL)
hESC-derived neurons were fixed in 4% paraformaldehyde for 1 h at room temperature and then permeabilized in 0.2% Triton X-100/phosphate-buffered solution for 15 min. TUNEL staining was performed according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, POD; Roche). Stained cells were analyzed using the BD FACSAria Cell Sorter.