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An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material

机译：一种优化方案，用于通过激光捕获微量叶材料分离高质量RNA

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摘要

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low-melting-point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high-quality RNA suitable for down-stream applications such as quantitative reverse transcriptase-polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.
机译：激光捕获微量碎石是一种强大的工具，允许薄片的特定小区类型彼此分开。然而，最常用的方案涉及在石蜡中嵌入组织，导致严重降解的RNA。来自低丰度细胞类型的叶片的产量特别受到损害。我们推理用于样品嵌入的相对较高的温度，以及在微粉切除之前与样品制备相关的水性条件有助于RNA降解。在此，我们描述了限制基于低熔点蜡的RNA劣化的优化程序以及在解剖之前对样品制备的修饰，以及分离帕拉索尔，而不是横截面。使用这种方法，从微小束鞘股和叶组织的微小束鞘股和叶片细胞中回收适用于诸如定量逆转录酶 - 聚合酶链反应或RNA测序的高质量RNA。

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