Tissue extraction kit

In total, 15-20 retinal organoids were homogenised using a Dounce Tissue Grinder (Sigma-Aldrich, UK) and RNA was extracted using the Promega tissue extraction kit (Promega, USA) as per manufacturer’s instructions. In all, 1 μg of RNA was reverse transcribed using random primers (Promega, USA). qRT-PCR was performed using a Quant Studio 7 Flex system (Applied Biosystems, USA) with SYBR Green (Promega, USA). Each primer (Table S2) was used at a concentration of 1 μM, and at a ratio of 50:50 for forward and reverse. The reaction parameters were as follows: 95 °C for 15 minutes to denature the complementary DNA and primers, 40 cycles of 94 °C for 15 seconds followed by primer specific annealing temperature for 30 seconds, succeeded by a melt curve. The data were analysed using 2-ΔΔCt method. Statistical analysis was done using Prism 6 (GraphPad Software, La Jolla, CA). All results were validated using Student’s t-test for paired samples.

About 15–20 retinal organoids were homogenized using a Dounce Tissue Grinder (Sigma) and RNA was extracted using the Promega tissue extraction kit (Promega, Madison, WI) as per the manufactures instructions. About 1 μg of RNA was reverse transcribed using random primers (Promega). qRT‐PCR was performed using a Quant Studio 7 Flex system (Applied Biosystems, Foster City, CA) with SYBR Green (Promega). Each primer (listed in Supporting Information Table S1) was used at a concentration of 1 μM, and at a ratio of 50:50 for forward and reverse. The reaction parameters were as follows: 95°C for 15 minutes to denature the cDNA and primers, 40 cycles of 94°C for 15 seconds followed by primer specific annealing temperature for 30 seconds, succeeded by a melt curve. All primer pairs were validated on adult human retina. A comparative Ct method was used to calculate the levels of relative expression, whereby the Ct was normalized to the endogenous control (GAPDH). This calculation gives the ΔCt value, which was then normalized to WT1, giving the ΔΔCt. The fold change was calculated using the following formula: 2^−ΔΔCt. Statistical significance analyses were evaluated using Prism (GraphPad, La Jolla, CA).