Cox Melissa L, Eddy Susan M, Stewart Zachary S, Kennel Maggi R, Man Michael Z, Paulauskis Joseph D, Dunstan Robert W
Drug Safety, Pfizer Global Research and Development, Ann Arbor, MI, USA.
Exp Mol Pathol. 2008 Apr;84(2):156-72. doi: 10.1016/j.yexmp.2007.11.002. Epub 2007 Dec 3.
Described herein is a detailed analysis of the impact of three fixatives (10% neutral buffered formalin, modified methacarn and 70% ethanol) on RNA quality and utility using microarray analysis compared to OCT-embedded and flash frozen tissue. From rat livers fixed and stored in paraffin blocks for 1 month or 1 year, RNA was isolated and applied to rat whole genome microarrays. At both time points, RNA isolated from OCT-embedded tissue lost up to 5% of the information contained in snap frozen control liver. Of the fixatives used, modified methacarn was associated with the smallest loss of RNA information content (approximately 10%), while liver fixed in 70% ethanol and 10% neutral buffered formalin lost roughly 25% and 80%, respectively. We conclude that when optimum morphology is required for techniques such as laser microdissection, modified methacarn is the fixative least harmful to nucleic acids of the three tested in this study. In contrast, using traditional isolation techniques, RNA derived from tissue fixed in 10% NBF will not give reliable results on microarray studies, and should be reserved for techniques less affected by the fragmentation and modification of the template RNA, such as quantitative RT-PCR.
本文描述了与OCT包埋和速冻组织相比，使用微阵列分析对三种固定剂（10%中性缓冲福尔马林、改良甲醇 Carnoy 固定液和 70%乙醇）对RNA质量和实用性的影响进行的详细分析。从固定并储存在石蜡块中1个月或1年的大鼠肝脏中分离RNA，并将其应用于大鼠全基因组微阵列。在这两个时间点，从OCT包埋组织中分离的RNA损失了速冻对照肝脏中所含信息的5%。在所使用的固定剂中，改良甲醇 Carnoy 固定液导致的RNA信息含量损失最小（约10%），而用70%乙醇和10%中性缓冲福尔马林固定的肝脏分别损失约25%和80%。我们得出结论，当激光显微切割等技术需要最佳形态时，改良甲醇 Carnoy 固定液是本研究中测试的三种固定剂中对核酸危害最小的固定剂。相比之下，使用传统分离技术，来自10%中性缓冲福尔马林固定组织的RNA在微阵列研究中不会给出可靠结果，应保留用于受模板RNA片段化和修饰影响较小的技术，如定量RT-PCR。
