Quantifying Eye and Retinal Development

To confirm the observed developmental changes, we evaluated the expression of several eye- and retina-specific marker genes (Supplemental Fig. S1b). Retinal and anterior neural fold homeobox (RAX) are expressed early in the eye primordia and are essential for retinal development^{16 (link)}. PAX6 is essential for eye development and a candidate gene for congenital aniridia^{17 (link)}. POU4F2/BRN3B is expressed in RGCs^{18 (link)} and suppresses the differentiation of other types of retinal cells. Total RNA was isolated from three to six EBs on days 3, 10, 17, 25, and 35 using an RNeasy microRNA isolation kit (QIAGEN, Japan). Isolated RNAs were treated with reverse transcriptase (TaKaRa, Ohtsu, Japan) to generate cDNA. The expression of marker genes of eyes and retinal development (RAX, PAX6, and POU4F2) was assessed using real-time qPCR with the following primers:

RAX forwards: CCCTAAGCGTGCTTTCAGGA

RAX reverse: TTTGCTCAGGACCGACAGAC

PAX6 forwards: TGGCTCACCAAGGCGAAATA

PAX6 reverse: CCGAGCAGTTGAGTCATTCAG

POU4F2 forwards: GAACGAGCGAACAACTGAGC

POU4F2 reverse: CTCTGAAGAAGCCGAGGTGG

SCS (18S) forwards: TGTGTGAAAGATTAAGCATGCA

SCS (18S) reverse: GCGACCAAAGGAACCATAACTG

We used Premix Ex Taq (Perfect Real-Time, Takara bio) and a CFX Connect Real-Time PCR Detection System (Bio-Rad, Japan). The results were normalized to SCS (18S) expression. All experiments were run in triplicate (Supplemental Fig. S1b).

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Katsura M., Urade Y., Nansai H., Kobayashi M., Taguchi A., Ishikawa Y., Ito T., Fukunaga H., Tozawa H., Chikaoka Y., Nakaki R., Echigo A., Kohro T., Sone H, & Wada Y. (2023). Low-dose radiation induces unstable gene expression in developing human iPSC-derived retinal ganglion organoids. Scientific Reports, 13, 12888.

Publication 2023

RNeasy microRNA isolation kit reverse transcriptase Premix Ex Taq (Perfect Real-Time, CFX Connect Real-Time PCR Detection System

A gene Cdna Cells types differentiation Expression genes Eyes Genes Homeobox Microrna Neural fold Primers Retinal Reverse transcriptase Rnas

Corresponding Organization : Yokohama University of Pharmacy

Other organizations : Minamiaoyama Eye Clinic, Jichi Medical University

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Variable analysis

independent variables

Days of embryoid body (EB) development (3, 10, 17, 25, and 35)

dependent variables

Expression of eye- and retina-specific marker genes (RAX, PAX6, and POU4F2)

control variables

Expression of SCS (18S) reference gene

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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