Foss R D, Guha-Thakurta N, Conran R M, Gutman P
Oral Pathology Department, Naval Dental School, National Naval Dental Center, Bethesda, MD 20889-5602.
Diagn Mol Pathol. 1994 Sep;3(3):148-55. doi: 10.1097/00019606-199409000-00003.
A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2, 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II, in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT, DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues.
许多报告表明，从石蜡包埋组织中回收的RNA可作为聚合酶链反应（PCR）的模板。尽管已确定石蜡包埋组织中的RNA会发生显著降解，但不同固定剂和固定时间对这种降解的具体影响尚不清楚。采集小鼠脾脏组织，立即用福尔马林、Omnifix II或卡诺固定液固定2、8或24小时，然后进行处理并包埋在石蜡中。使用Chomczynski和Sacchi描述的技术改进方法，从脱蜡的组织块中提取RNA。使用随机六聚体引发反应对RNA进行逆转录。然后对管家基因甘油醛-3-磷酸脱氢酶（GAPDH）和次黄嘌呤磷酸核糖转移酶（HPRT）mRNA的cDNA进行PCR扩增。尽管GAPDH扩增常用于新鲜和冷冻组织，但我们发现RNA制剂中存在的DNA污染限制了其在石蜡包埋组织中的应用。在12个用Omnifix II固定的样本中有9个检测到可扩增的HPRT mRNA序列，在12个用卡诺固定液固定的样本中有4个检测到，而在12个福尔马林固定的样本中均未检测到。由于引物选择可排除基因组HPRT的扩增，因此扩增HPRT时不存在DNA污染问题。因此，HPRT是评估石蜡包埋组织中RNA的首选对照系统。所描述的技术提供了一种从石蜡包埋组织中获取RNA进行分子分析的快速、统一且可重复的方法，但它们表明对存档组织进行回顾性分析的实用性有限。
