Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality
Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality
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DOI：
10.1016/j.yexmp.2012.07.002
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发表时间：
2013-02-01
影响因子：
3.6
通讯作者：
Rainen, Lynne
中科院分区：
文献类型：
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作者：
Groelz, Daniel;Sobin, Leslie;Rainen, Lynne
组织样本的分析前处理会影响生物分析物的质量和分析结果的最终结果。本研究的目的是在高度受控的分析前条件下，在动物模型中比较福尔马林固定组织与用无福尔马林固定剂PAXgene组织系统（PAXgene）固定和稳定的组织的RNA质量、真实的时间RT PCR性能和组织学。将大鼠肝、肾、脾、肠、肺、心肌、脑和胃组织样品固定在福尔马林中或固定在PAXgene中或新鲜冷冻在液氮中。从所有样品中提取RNA，在微毛细管电泳中检查其完整性，并用于一系列扩增子长度增加的定量RT PCR测定。石蜡包埋标本的组织学通过苏木精和伊红染色来确定。所有福尔马林固定和PAXgene固定的标本的组织学具有可比性。从所有包埋的组织中可以分离出具有可接受的完整性评分的RNA，福尔马林为4.0至7.2，PAXgene为6.4至7.7，而新鲜冷冻样品为8.0至9.2。虽然可以从福尔马林固定的样品中分离出具有可接受的RINs（RNA完整性编号）的RNA，但与冷冻或PAXgene固定的样品中的RNA相比，在微毛细管电泳中，这种RNA以较慢的迁移速率分离，并显示出弥散的、不太集中的核糖体RNA峰。此外，RNA从福尔马林固定的组织表现出抑制在定量RI PCR检测增加扩增子的长度，而RNA从PAXgene固定的样品没有表现出这样的inhibit1n总之，我们的研究结果表明，排除其他分析前的因素，PAXgene组织系统保留组织类似于福尔马林，但不像福尔马林，不化学修饰RNA。从PAXgene固定的组织中纯化的RNA具有高度完整性，并且无论扩增子长度如何，在RI PCR中与来自新鲜冷冻组织的RNA一样好。(C)2012 Elsevier Inc. All rights reserved.
Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin.Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RI PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition.In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RI PCR regardless of amplicon length. (C) 2012 Elsevier Inc. All rights reserved.