In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

中文翻译：

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快速，高效，高通量的过程，涉及激光显微切割和RT液滴数字PCR，用于水稻根的组织特异性表达谱分析

在水稻中，皮层和外部组织在淹没耐受性中起关键作用。皮质分化为气孔，气孔是空气中的空洞，使氧气从枝条流向根部，而外皮亚铁化作用和巩膜木质化通过形成对根氧损失（ROL）的屏障来限制根部成熟部分的氧损失。与这些组织的细胞身份和分化有关的基因及其网络仍然知之甚少。鉴定和表征通气组织和ROL屏障形成的关键调节剂需要确定这些组织的特异性表达谱。我们优化了结合激光显微切割（LM）和液滴数字RT-PCR（ddRT-PCR）的方法，以高通量鉴定组织特异性表达谱。所开发的方案可以从污染低的根组织中快速（3天之内）提取高质量的RNA。我们还证明了从储存在4°C的石蜡块中提取RNA而不损失质量的可能性。我们提供了一份详细的故障排除指南，该指南应允许将来的用户使建议的协议适应其他组织和/或物种。我们证明，将LM与ddRT-PCR结合使用的方案可以用作原位杂交的补充工具，以用于基因表达的组织特异性表征，即使输入的RNA浓度较低。我们通过验证RiceXpro数据库中详述的四个潜在组织特异性候选基因中的三个来说明了所提出方法的效率。