Laser capture microdissection for transcriptomic profiles in human skin biopsies

IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology BMC Molecular Biology Pub Date : 2018-06-19 DOI:10.1186/s12867-018-0108-5
Silvia Santoro, Ignazio Diego Lopez, Raffaella Lombardi, Andrea Zauli, Ana Maria Osiceanu, Melissa Sorosina, Ferdinando Clarelli, Silvia Peroni, Daniele Cazzato, Margherita Marchi, Angelo Quattrini, Giancarlo Comi, Raffaele Adolfo Calogero, Giuseppe Lauria, Filippo Martinelli Boneschi
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引用次数: 11

Abstract

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.

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激光捕获显微解剖的转录组谱在人体皮肤活检
从人体皮肤活检中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,通过激光捕获显微解剖从新鲜冷冻人体标本中分离特定层的过程的复杂性,皮肤核酸酶的丰富存在和RNA的不稳定性仍然是相关的方法学挑战。我们开发并优化了一种从人体皮肤活检层中提取RNA的方案,并提供了令人满意的质量和数量的mRNA测序数据。该方案包括收集、包埋、冷冻、组织染色和相对优化的步骤,以保存从14名受试者的新鲜冷冻人体皮肤活检的特定成分中提取的RNA。协议的优化包括RNALater?溶液,样品温度的控制,RNase抑制剂的使用和染色程序的时间减少。用长度超过200个核苷酸的片段百分比(DV200)来测量提取的RNA的质量,这比RNA完整性数(RIN)更适合于成功的文库制备。然后用TruSeq?RNA Access Library Prep Kit (Illumina?)并在HiSeq??2500平台(Illumina?)对RNA测序数据的质量控制足以为下游分析提供可靠的数据。所述的实现和优化的方案可用于在皮肤组织上生成转录组学数据,并且它可能适用于其他组织。它可以扩展到多中心研究,因为引入了保存标本的初始步骤,允许运送生物样品。
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来源期刊
BMC Molecular Biology
BMC Molecular Biology 生物-生化与分子生物学
CiteScore
4.80
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: BMC Molecular Biology is an open access journal publishing original peer-reviewed research articles in all aspects of DNA and RNA in a cellular context, encompassing investigations of chromatin, replication, recombination, mutation, repair, transcription, translation and RNA processing and function.
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