Kap Marcel, Sieuwerts Anieta M, Kubista Mikael, Oomen Monique, Arshad Shazia, Riegman Peter
1Erasmus MC Tissuebank and Tissue Research Support Unit, Department of Pathology, Erasmus University Medical Centre, Rotterdam, The Netherlands.
2Department of Medical Oncology, Erasmus MC Cancer Institute, Cancer Genomics Netherlands, Erasmus University Medical Centre, Rotterdam, The Netherlands.
Biopreserv Biobank. 2015 Jun;13(3):200-6. doi: 10.1089/bio.2014.0076.
The advent of molecular characterization of tissues has brought an increasing emphasis on the quality of biospecimens, starting with the tissue procurement process. RNA levels are particularly affected by factors in the collection process, but the influence of different pre-analytical factors is not well understood. Here we present the influence of tissue specimen size, as well as the transport and freezing protocols, on RNA quality. Large, medium, and smaller porcine liver samples were stored either dry, on moist gauze, or in salt solution for various times, and then frozen in either liquid nitrogen or in pre-cooled isopentane. Large and small human liver samples were frozen in pre-cooled isopentane either immediately or after one hour at room temperature. The small samples were stored dry, on moist gauze, or in salt solution. RNA was isolated and RIN values were measured. The RNA for six standard reference genes from human liver was analyzed by RT-qPCR, and tissue morphology was assessed for artifacts of freezing. Experiments using porcine liver samples showed that RNA derived from smaller samples was more degraded after one hour of cold ischemia, and that cooled transport is preferable. Human liver samples showed significant RNA degradation after 1 h of cold ischemia, which was more pronounced in smaller samples. RNA integrity was not significantly influenced by the transport or freezing method, but changes in gene expression were observed in samples either transported on gauze or in salt solution. Based on observations in liver samples, smaller samples are more subject to gene expression variability introduced by post-excision sample handling than are larger samples. Small biopsies should be transported on ice and snap frozen as soon as possible after acquisition from the patient.
组织分子特征分析的出现使得人们越来越重视生物样本的质量，这始于组织采集过程。RNA水平特别受采集过程中各种因素的影响，但不同分析前因素的影响尚未得到充分了解。在此，我们展示了组织样本大小以及运输和冷冻方案对RNA质量的影响。将大、中、小不同尺寸的猪肝样本分别干燥保存、置于湿纱布上保存或置于盐溶液中保存不同时间，然后在液氮或预冷的异戊烷中冷冻。大、小尺寸的人肝样本立即或在室温下放置一小时后在预冷的异戊烷中冷冻。小样本分别干燥保存、置于湿纱布上保存或置于盐溶液中保存。提取RNA并测量RIN值。通过RT-qPCR分析来自人肝的六个标准参考基因的RNA，并评估组织形态以检查冷冻假象。使用猪肝样本的实验表明，较小样本来源的RNA在冷缺血一小时后降解更严重，因此冷却运输更可取。人肝样本在冷缺血1小时后显示出明显的RNA降解，在较小样本中更为明显。RNA完整性受运输或冷冻方法的影响不显著，但在置于纱布或盐溶液中运输的样本中观察到基因表达变化。基于对肝样本的观察，与大样本相比，较小样本在切除后样本处理过程中更容易受到基因表达变异性的影响。小活检样本应在从患者获取后立即在冰上运输并尽快速冻。
