Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Division of Experimental Retinal Therapies, Department of Clinical Sciences & Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Exp Eye Res. 2024 Sep;246:109989. doi: 10.1016/j.exer.2024.109989. Epub 2024 Jul 3.
RNA sequencing (RNA-seq) coupled with laser capture microdissection (LCM) is a powerful tool for transcriptomic analysis in unfixed fresh-frozen tissues. Fixation of ocular tissues for immunohistochemistry commonly involves the use of paraformaldehyde (PFA) followed by embedding in Optimal Cutting Temperature (OCT) medium for long-term cryopreservation. However, the quality of RNA derived from such archival PFA-fixed/OCT-embedded samples is often compromised, limiting its suitability for transcriptomic studies. In this study, we aimed to develop a methodology to extract high-quality RNA from PFA-fixed canine eyes by utilizing LCM to isolate retinal tissue. We demonstrate the efficacy of an optimized LCM and RNA purification protocol for transcriptomic profiling of PFA-fixed retinal specimens. We compared four pairs of canine retinal tissues, where one eye was subjected to PFA-fixation prior to OCT embedding, while the contralateral eye was embedded fresh frozen (FF) in OCT without fixation. Since the RNA obtained from PFA-fixed retinas were contaminated with genomic DNA, we employed two rounds of DNase I treatment to obtain RNA suitable for RNA-seq. Notably, the quality of sequencing reads and gene sets identified from both PFA-fixed and FF tissues were nearly identical. In summary, our study introduces an optimized workflow for transcriptomic profiling from PFA-fixed archival retina. This refined methodology paves the way for improved transcriptomic analysis of preserved ocular tissue, bridging the gap between optimal sample preservation and high-quality RNA data acquisition.
RNA 测序（RNA-seq）与激光捕获显微切割（LCM）相结合，是分析未经固定的新鲜冷冻组织中转录组的有力工具。为了进行免疫组织化学染色，眼部组织的固定通常涉及使用多聚甲醛（PFA），然后嵌入最佳切割温度（OCT）介质中进行长期冷冻保存。然而，从这种存档的 PFA 固定/OCT 嵌入样品中获得的 RNA 质量往往会受到影响，限制了其在转录组研究中的适用性。在这项研究中，我们旨在开发一种利用 LCM 从 PFA 固定的犬眼组织中提取高质量 RNA 的方法。我们展示了一种优化的 LCM 和 RNA 纯化方案，用于对 PFA 固定的视网膜标本进行转录组分析。我们比较了四对犬视网膜组织，其中一只眼在 OCT 嵌入前接受 PFA 固定，而对侧眼则在 OCT 中新鲜冷冻（FF）嵌入而不固定。由于从 PFA 固定的视网膜中获得的 RNA 被基因组 DNA 污染，我们采用两轮 DNase I 处理来获得适合 RNA-seq 的 RNA。值得注意的是，从 PFA 固定和 FF 组织中获得的测序读数和基因集的质量几乎相同。总之，我们的研究介绍了一种从 PFA 固定的存档视网膜进行转录组分析的优化工作流程。这种改进的方法为保存的眼部组织的转录组分析铺平了道路，弥合了最佳样本保存和高质量 RNA 数据获取之间的差距。
