Cfx light cycler

Total RNA was isolated from Arabidopsis leaves using the Monarch Total RNA Miniprep Kit including the on-column DNase digest, and cDNA was synthesized according to the instructions supplied with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, United States). For quantitative real-time (qRT) PCR, SYBR Green Supermix (Bio-Rad) was used and PCR was performed with the CFX light cycler (Bio-Rad). Selected regions of the cTP_(PAM71) sequence (primers 30/31) or the PAM71_(core) sequence (primers 32/33) were used to record expression of the target gene and the reference gene Actin (primers 28/29). Relative transcript levels were quantified according to the comparative cycle threshold (C_T) method (Schmittgen and Livak, 2008 (link)): ΔC_T = C_T (Gene_{TransgenicLine}) − C_T (Actin_{TransgenicLine}) or ΔC_T = C_T (Gene_Col–0) − C_T (Actin_Col–0); ΔΔC_T (TransgenicLine) = ΔC_T (Gene_{TransgenicLine}) − ΔC_T (Gene_Col–0) and 2^–ΔΔCT calculated. Expression in Col-0 is: ΔΔC_T (Col-0) = ΔC_T (Gene_Col–0) − ΔC_T (Gene_Col–0) = 0 and 2⁰ = 1.

Cells were differentiated according to the protocol described by Hazim et al. (20 (link)). Briefly, cells were cultivated in T75 flask for two weeks with MEM-nicotinamide medium: 1% N1 (Invitrogen), 1% SVFi, 1% P/S, 0.25mg/mL Taurine (Sigma-Aldrich), Hydrocortisone 20ng/mL (Sigma-Aldrich), Triiodo-thyronine 0.013 ng/mL (Sigma-Aldrich) and 10mM nicotinamide (Sigma-Aldrich). After 3 times 10min TrypLE (Gibco) treatment, the remaining cells were placed on 0.4µM PET transwell inserts (Falcon, 353095) in 24-well-plates, coated with natural mouse laminin (Invitrogen). Cell medium was changed 3 times per week during 8 weeks before use. Differentiation was verified using RT-PCR analysis of markers indicating primary like cell phenotype. Cells on transwells were harvested every week during 8 weeks using Nucleozol (Macherey-Nagel, MN). RNA was extracted with TriZol reagent, according to the manufacturer’s recommendations. Then, 20ng of RNA was reverse transcribed to cDNA using Qscript reagent (VWR). Real-time PCR specific for the BEST1, RPE65, OCLN, MERTK, ZO-1, ITGB5 and housekeeping gene GAPDH mRNA (Table 1) was performed on CFX light cycler and SSoAdvanced SybrGreen (Biorad).