PDF(Pubmed)
Abstract：
Background: Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts. Methods: Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq. Results: The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%. Conclusions: Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples.
摘要：
背景：由于该组织的物理组成和高含量的核酸酶，从皮肤活检中获得高质量的RNA是复杂的。当使用坏死的受损皮肤样本时，这变得特别具有挑战性，发炎或受损区域，比如那些患有皮肤病的病人，每年影响超过9亿人。我们评估了活检大小和组织保存方法对RNA提取物质量和数量的影响。方法：从皮肤利什曼病（CL）患者中获得皮肤病变活检。将2mm（n=10）和3mm（n=59）的活检标本保存在Allprotect®试剂中，和OCT中的4mm活检(n=54)。使用Nanodrop和Bioanalyzer评估质量参数。使用RT-qPCR和RNA-Seq评估用于下游分析的提取样品的信息量。结果：成功率，根据储存在OCT中的组织活检和储存在Allprotect®中的2mm活检的RNA提取质量参数，分别为56%(30/54)和30%(3/10)，分别。对于储存在Allprotect®中的3mm皮肤活检为93%(55/59)。来自3mm-Allprotect®活检的RNA制剂具有7.2±0.7的平均RIN,并且它们的完整性不受样品储存时间(在-20°C下长达200天)的影响。RNA产物适用于qRT-PCR和RNA-seq。基于这些结果,我们提出了一种从破裂的皮肤样本中提取RNA的标准化方法。该方案通过CL患者（n=30）的病变活检进行了验证，成功率为100%。结论：我们的结果表明，直径为3mm的活检大小，并在Allprotect®中在-20°C下保存长达200天，最好从溃疡皮肤病变活检样品中获得高质量的RNA制剂。
