A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection
A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection
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DOI：
10.1007/s10265-010-0319-4
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发表时间：
2010-11-01
影响因子：
2.8
通讯作者：
Nakazono, Mikio
中科院分区：
文献类型：
--
作者：
Takahashi, Hirokazu;Kamakura, Hisae;Nakazono, Mikio
激光显微切割（LM）结合微阵列分析或下一代cDNA测序是理解植物和动物个体细胞类型中分子事件的有力工具。获得高质量的RNA对于这种方法至关重要。对于植物组织，石蜡包埋切片比冷冻切片更好地保存细胞结构。然而，制备石蜡切片的常规方法是一个冗长的过程，涉及包埋组织和漂浮并干燥切片，在此期间发生RNA降解。本文介绍了一种能大大减少RNA降解的连续切片制备方法：（1）微波法将包埋时间从4-6天缩短到5小时左右，（2）漂浮时间从10分钟左右缩短到5分钟以下，（3）干燥时间从12分钟左右缩短到1小时;干燥温度为42 ~ 4A ℃。利用这种方法，我们能够从许多种植物组织中分离出比通常通过常规石蜡制备方法获得的更高完整性的RNA。RNA质量和产量的提高消除了LM与植物高通量技术广泛使用的主要障碍。
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 similar to 10 min to less than 5 min, (3) the drying time from similar to 12 to 1 h; and (4) the drying temperature from 42 to 4A degrees 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.