RNA extraction and RT-qPCR

In each experimental repeat, 15 pairs of eye discs were homogenized with 0.3 mL TRIzol (Invitrogen, 15596026), and total RNA was extracted by following the product manual. For RT-qPCR experiment, 1 μg total RNA was used for cDNA synthesis with Taqman Reverse Transcription Reagents (Applied Biosystems, N8080234). After 1:3 dilution, the cDNA was used as a template to perform qPCR with SYBR Green PCR Master Mix (Applied Biosystems, 4309155). The qPCR was run on a Quantstudio5 machine (Applied Biosystems). The fold changes of a gene in different samples are calculated by 2 to the power of ΔCt. The expression of housekeeping gene rp49 was used to normalize the expression of tested genes. P-values of relative levels between samples were calculated by T.TEST with one-tailed distribution and paired type.