大麻中腺头柄和无柄毛状体的非水分离和富集
Summary
提出了一种方案，用于方便和高通量地从 大麻中分离和富集腺头柄和无柄毛状体。该方案基于仅使用液氮、干冰和尼龙筛对毛状体进行干燥、非缓冲提取，适用于 RNA 提取和转录组学分析。
Abstract
本文提出了一种方便、高通量地从 大麻中分离和富集腺头柄和无柄毛状体的方案。大麻素和挥发性萜烯代谢的生物合成途径主要位于 大麻 毛状体中，分离的毛状体有利于转录组分析。用于分离腺毛以进行转录组学表征的现有方案不方便，并且提供受损的毛状体头和相对较少数量的分离毛状体。此外，它们依靠昂贵的设备和含有蛋白质抑制剂的分离培养基来避免RNA降解。本方案建议结合三种单独的修饰，分别从 苜蓿 成熟的雌性花序和扇形叶中获得大量分离的腺头柄和无柄毛状体。第一种修改涉及用液氮代替传统的隔离介质，以促进毛状体通过微筛。第二种修改涉及使用干冰将毛状体与植物源头分离。第三种修改涉及将植物材料连续通过五个孔径减小的微筛。显微成像证明了分离技术对两种毛状体类型的有效性。此外，从分离的毛状体中提取的RNA质量适合下游转录组分析。
Introduction
腺毛是存在于植物中的毛发状结构，含有许多次级代谢物1，代表了新型生物合成基因和酶的宝贵库2。在大麻中，重要的次生代谢物大麻素3和萜烯4的生物合成位于毛状体中。考虑到毛状体在确定药用和娱乐用途大麻质量方面的作用，毛状体基因表达的研究是有意义的。为了表征毛状体特异性基因的表达，必须首先分离感兴趣的毛状体。毛状体分离方案早在1992年就被首次描述5，其最新进展最近已得到回顾2。通常，提取腺毛状体以进行转录组学表征的方案可分为两个不同的顺序步骤。第一步涉及毛状体与植物组织的彻底物理分离。该步骤可以通过使用干冰5、玻璃珠与商业设备6、7、在网筛8上研磨植物材料、或在隔离缓冲液9中涡旋植物组织来执行。第二步涉及将感兴趣的毛状体与微观植物残留物和/或其他毛状体类型进行更精细的分离。该步骤可以使用密度梯度离心8，10或各种尺寸的筛子7，9执行。由于加工组织中的RNA对降解剂的敏感性极高，这两个连续步骤通常在冰冷的分离介质中进行，通常在蛋白质抑制剂4的存在下进行。
除了冰冷的温度外，传统的毛状体分离方案还需要大量的分离介质，以确保有效的提取过程。这些组件的组合导致一个艰巨、耗时的隔离过程，阻碍了高吞吐量。因此，提出一种简单、用户友好的替代毛状体分离方案可能对与毛状体表征相关的各个方面有益。本文旨在通过结合和整合传统方案中的几种元素，提供一种替代方案，从大麻中分离出柄状状体和无柄腺头状体。这些元素包括干冰5，毛状体通过几个孔径减小的微筛7，9，以及用液氮（LN）代替隔离介质8。
与传统方案相比，目前的毛状体分离方案的新颖性以多种方式呈现。该协议很方便，因为它不需要危险成分。该程序可以在实验室中进行，只需最少的预防措施，并促进高通量。用LN代替标准液体分离介质可确保毛状体在整个分离过程中的完整性，从而实现后续的转录组学分析。LN和干冰升华后，分离的毛状体没有有害残留物。此外，LN在室温下升华的倾向允许其在整个协议中大量使用。相反，使用大量常规隔离介质会在处理时产生实际困难。最后，该方案减少了椎间盘细胞与腺毛体剩余脆弱头部结构的分离，从而能够保留顶空内容物。
该协议以详细的分步方式呈现，旨在帮助分离 苜蓿衣原 体腺头状体的技术实践。该协议提供了一个可管理的工作流程，可分离出具有高浓度和纯度的毛状体，适用于下游分子分析。
Protocol
Representative Results
Discussion
与目前可用的毛状体隔离方案相比，本协议描述了两种主要的修改。其中包括在初始步骤中使用干冰将毛状体从植物材料中分离出来，并用LN代替常用的液体缓冲介质。 C. sativa 毛状体净化的第一个修改是基于早期的方案，该协议引入了使用碎干冰将毛状体从天竺葵花梗分离5.虽然传统的毛状体分离方案通常使用小（50 mL）试管，但本研究使用1 L玻璃烧杯，其中包含大量碎?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢CannabiVar Ltd.的财政支持。所有植物材料均由以色列火山中心的Hinanit Koltai教授慷慨提供。
Materials
| Bioanalyzer RNA Pico 6000 chip | Agilent, Germany | Reorder number 5067-1513 | Lab-on-a-chip system |
| Transsonic-310 | Elma, Germany | D-78224 | Ultrasonic cleaning unit |
| TruSeq RNA Sample Prep Kit v2 | Illumina, USA | RS-122-2001 | Sample preperation for RNA sequencing library |
| Spectrum Plant Total RNA Kit | SIGMA-ALDRICH, USA | STRN50-1KT | Plant Total RNA Kit |
| Nylon micro-sieve with a mesh size of 350 µm (40 x 40 cm or larger than the circumference of the flour sifter) | Sinun Tech, Israel | r0350n350210 | Nylon screen aperture |
| Nylon micro-sieve with mesh size of 150 µm (size of 30 x 30 cm) | Sinun Tech, Israel | r0150n360465 | Nylon screen aperture |
| Nylon micro-sieve with mesh size o 105 µm (size of 30 x 30 cm) | Sinun Tech, Israel | r0105n320718 | Nylon screen aperture |
| Nylon micro-sieve with mesh size o 80 µm (size of 30 x 30 cm) | Sinun Tech, Israel | r0080n370465 | Nylon screen aperture |
| Nylon micro-sieve with mesh size o 65 µm (size of 30 x 30 cm) | Sinun Tech, Israel | r0065n340715 | Nylon screen aperture |
| Nylon micro-sieve with mesh size o 50 µm (size of 30 x 30 cm) | Sinun Tech, Israel | r0080n370465 | Nylon screen aperture |
| Up to 10 g of frozen plant material (stored in -80 oC or liquid nitrogen) | |||
| Suitable gloves for handling low temperatures | |||
| Safety goggles | |||
| 1 mm screen door (mosquito) mesh (strip of 30 x 100 cm) | |||
| Large strainer (colander) with holes approximately 5 mm | |||
| 1 L glass beaker | |||
| 1 block of dry ice (0.5-1 kg) | |||
| Hammer and hard flat object | |||
| Two 5 L plastic containers | |||
| Rubber bands | |||
| Large flour sifter or sieve strainer- preferably one with a detachable plastic ring on the circumference | |||
| Several large and small round bottom stainless steel containers. One of them should be larger than the flour sifter's circumference (approximately 40 cm in diameter), to minimize the loss of the sifted mass outside the round bottome stainless steel container | |||
| Pre-chilled (via liquid nitrogen) stainless steel spoon, spatula, and scoopula | |||
| Clean plate | |||
| Several clothespins | |||
| Pre-chilled (via liquid nitrogen) labeled 1.5 mL tubes with holes poked on the lid with a sterile needle | |||
| Two containers of liquid nitrogen | |||
| 1 cm wide painting brush |
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