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首页> 外文期刊>Journal of Plant Research >A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection
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A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection

机译：一种通过激光显微切割从植物组织石蜡切片中获得高质量RNA的方法

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摘要

Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4-6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.
机译：激光显微解剖（LM）与微阵列分析或cDNA的下一代测序相结合是了解植物和动物个体细胞类型中分子事件的强大工具。获得高质量的RNA对于这种方法至关重要。对于植物组织，石蜡包埋的切片比冷冻切片能更好地保存细胞结构。然而，用于制备石蜡切片的常规方法是一个漫长的过程，涉及包埋组织并漂浮和干燥切片，在此期间发生RNA降解。在这里，我们描述了一种制备连续切片的方法，该方法可以大大减少RNA降解：通过使用最新开发的微波方法，我们将（1）嵌入时间从4-6天减少到了大约5小时; （2）浮动切片的时间从〜10分钟到少于5分钟;（3）干燥时间从〜12到1小时; （4）干燥温度为42至4℃。通过这种方法，我们能够从许多植物组织中分离出比常规石蜡制备方法通常获得的更高完整性的RNA。 RNA质量和产量的提高消除了将LM与高通量技术广泛应用于植物的主要障碍。

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