This paper describes experimental procedures for the dissociation of retinal cells of the zebrafish (Danio rerio) for subsequent fluorescence-activated cell sorting (FACS) and gene expression studies. Methods for dissociation of zebrafish retinas followed by FACS and RNA isolation were optimized. This methodology was applied to isolate pure sorted samples of rods, long wavelength-sensitive (LWS) cones, medium wavelength-sensitive (MWS; RH2-2) cones, short wavelength-sensitive (SWS2) cones, and UV-sensitive (SWS1) cones from retinas obtained at selective life-history stages of the zebrafish, and for some of these photoreceptors, following retinal regeneration. We also successfully separated lws1-expressing and lws2-expressing LWS cones from fish of a transgenic line in which lws1 is reported with green fluorescence protein (GFP) and lws2 is reported with red fluorescence protein (RFP). Microglia/macrophages were successfully sorted from regenerating retinas (7 days after a cytotoxic lesion) of a transgenic line in which these immune cells express GFP. Electropherograms verified downstream isolation of high-quality RNA from sorted samples. Examples of post-sorting analysis, as well as results of qRT-PCR studies, validated the purity of sorted populations. For example, qRT-PCR samples derived from isolated Rh2-2 cones contained detectable rh2-2 (opn1mw2) opsin transcripts, but lws opsin transcripts (lws1/opn1lw1, lws2/opn1lw2) were not detected, suggesting that the procedure likely separated double cone pairs. Through this method, pure, sorted cell samples can provide RNA that is reliable for downstream gene expression analyses, such as qRT-PCR and RNA-seq, which may reveal molecular signatures of photoreceptors and microglia for comparative transcriptomics studies.

本文介绍了用于分离斑马鱼视网膜细胞（Danio rerio）的实验程序，用于随后的荧光激活细胞分选（FACS）和基因表达研究。优化了解离斑马鱼视网膜，然后进行FACS和RNA分离的方法。该方法学用于分离棒，长波长敏感（LWS）圆锥，中波长敏感（MWS; RH2-2）圆锥，短波长敏感（SWS2）圆锥和紫外线敏感（SWS1）的纯分类样品视网膜再生后，在斑马鱼的选择性生活史阶段以及某些感光细胞中从视网膜获得的视锥细胞。我们还成功地分离了lws1 -expressing和lws2来自转基因鱼的表达LWS视锥细胞，其中lws1报道带有绿色荧光蛋白（GFP），lws2报道带有红色荧光蛋白（RFP）。从这些免疫细胞表达GFP的转基因系的再生视网膜（细胞毒性病变后7天）的视网膜中成功地分类了小胶质细胞/巨噬细胞。电泳图证实了从分类样品中分离出高质量的RNA。排序后分析的实例以及qRT-PCR研究的结果验证了排序后群体的纯度。例如，衍生自分离的Rh2-2视锥细胞的qRT-PCR样品包含可检测的rh2-2（opn1mw2）视蛋白转录本，而lws视蛋白转录本（未检测到lws1 / opn1lw1，lws2 / opn1lw2，这表明该过程可能会分离双锥对。通过这种方法，纯净的，经过分类的细胞样品可以提供可靠的RNA，用于下游基因表达分析，例如qRT-PCR和RNA-seq，这可以揭示感光细胞和小胶质细胞的分子特征，用于比较转录组学研究。