The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data. The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis. The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.

中文翻译：

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激光捕获显微解剖用于人类皮肤活检中的转录组谱。

从人皮肤活检获得可靠的组织特异性RNA测序数据代表了研究的重大进展。然而，通过激光捕获显微切割从新鲜冷冻的人类标本中分离特定层的过程的复杂性，皮肤核酸酶的大量存在以及RNA的不稳定性仍然是方法学上的挑战。我们开发并优化了从人体皮肤活检层中提取RNA并提供令人满意的质量和数量的mRNA测序数据的协议。该方案包括收集，嵌入，冷冻，组织学着色和相对优化的步骤，以保存从14名受试者的新鲜冷冻人皮肤活检组织的特定成分中提取的RNA。方案的优化包括RNALater®Solution中的保存步骤，样品温度的控制，RNase抑制剂的使用和减少染色程序的时间。使用大于200个核苷酸的片段百分比（DV200）测量提取的RNA的质量，比RNA完整性数（RIN）更适合成功进行文库制备。然后使用TruSeq®RNA访问文库制备试剂盒（Illumina®）富集RNA，并在2500平台（Illumina®）上测序。RNA测序数据的质量控制足以获得可靠的数据用于下游分析。所描述的已实现和优化的协议可用于在皮肤组织上生成转录组学数据，并且可能适用于其他组织。由于引入了标本保存的初始步骤，该步骤允许运输生物样品，因此可以扩展到多中心研究。