7500 fast real time pcr system apparatus

RNA were extracted from separated retina and RPE/Choroid according to the manufacturer’s protocol using 2 DNAse steps (Illustra RNAspin Mini, GE Healthcare, Vélizy-Villacoublay, France). RNAs were verified on 1% agarose gels and yields assessed using a spectrophotometer. Then, 500 ng of RNAs were converted to cDNAs in a 50-µL volume following the instructions provided for 1 hour at 42 °C (Reverse Transcription System, Promega, Charbonnières, France). qPCR reactions using the SYBR Green PCR Master Mix were processed as follows on a 7500 Fast Real-Time PCR System apparatus (both Applied Biosystems) using the primer pairs detailed in Supplementary Table S2: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec, 60 °C for 1 min. The ribosomal protein Rho0 (Rplp0) gene was used as internal control. Oligonucleotides were designed in order to obtain 150-bp amplicons and associated melting curves from a typical experiment series are pictured in Supplementary Figure S1. Relative amounts of each target gene were calculated using the 2^−ΔΔCt method and amounts at 8 AM (8.00, light onset) were set as 1.

RNA samples were isolated from both, iPS cells or iNeurons (DIV28) using Nucleo Spin RNA isolation kit (Machery Nagel, 740955.250) according to the manufacturer’s instructions. RNA samples (200 ng) were converted into cDNA by iScript cDNA synthesis kit (BIO-RAD, 1708891). cDNA products were cleaned up using the Nucleospin Gel and PCR clean-up kit (Machery Nagel, 740609.250). Human-specific primers were designed with Primer3plus and IDT PrimerQuest tools, respectively. Primer sequences are given in supplementary Table 1. qPCRs were performed in the 7500 Fast Real Time PCR System apparatus (Applied Biosystems) with GoTaq qPCR master mix 2× with SYBR Green (Promega, A600A) according to the manufacturer’s protocol. The PCR program was designed as following: After an initial denaturation step at 95 °C for 10 min, PCR amplifications proceeded for 40 cycles of 95 °C for 15 s and 60 °C for 30 s and followed by a melting curve. All samples were analyzed in duplicate in the same run, placed in adjacent wells. Reverse transcriptase-negative controls and no template-controls were included in all runs. The arithmetic mean of the Ct values of the technical replicas was used for calculations. Relative mRNA expression levels were calculated using the 2^−ΔΔCt method^{61 (link)} with standardization to housekeeping genes.