一种从多种植物中提取高质量 RNA 用于下一代测序和基因表达分析的方法。
A method for extracting high-quality RNA from diverse plants for next-generation sequencing and gene expression analyses.
机构信息
Department of Plant and Microbial Biology, Department of Integrative Biology, and the University and Jepson Herbaria, University of California, Berkeley, California 94720 USA ; Origine, Structure et Evolution de la Diversité (UMR 7205 CNRS), Muséum National d'Histoire Naturelle, CP39, 16 rue Buffon, 75231 Paris Cedex 05, France.
Department of Plant and Microbial Biology, Department of Integrative Biology, and the University and Jepson Herbaria, University of California, Berkeley, California 94720 USA.
出版信息
Appl Plant Sci. 2013 Dec 9;1(12). doi: 10.3732/apps.1300070. eCollection 2013 Dec.
PREMISE OF THE STUDY
To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNAlater. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues. •
METHODS AND RESULTS
Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNAlater or frozen fresh at -80°C. Extractions were performed and quality was measured for yield and purity. •
CONCLUSIONS
This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).
研究前提
为了研究植物中的基因表达，必须提取足够数量且质量高的 RNA 用于后续的 cDNA 文库构建。基于野外采集的样本通常在组织数量和质量上都存在限制，并且通常保存在 RNAlater 中。从各种保存的样本中获得足够且高质量的产量对于比较生物学的研究至关重要。我们提出了一种从最棘手的植物组织中提取高质量 RNA 的方案。
方法和结果
苔藓、苏铁和被子植物花器官及叶片组织保存在 RNAlater 或 -80°C 下冷冻新鲜。进行提取并测量产量和纯度以评估质量。
结论
该方案可从代表维管束和非维管束植物的各种植物组织中提取高质量的 RNA。RNA 用于 cDNA 合成，以生成用于下一代测序的文库，并用于使用定量 PCR(qPCR)和半定量反转录 PCR(RT-PCR)的表达研究。
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