Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction
Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction
复制标题
DOI：
10.1016/s1525-1578(10)60621-6
复制
发表时间：
2000-05-01
影响因子：
4.1
通讯作者：
Jensen, RH
中科院分区：
文献类型：
--
作者：
Godfrey, TE;Kim, SH;Jensen, RH
分析基因表达及其与临床参数的相关性有可能成为治疗决策的重要因素。分析存档组织中基因表达的能力，已经可以进行临床随访，将极大地促进这一领域的研究。然而，这种方法的一个主要障碍是对常规存档组织的基因表达分析是否准确反映固定前的表达的不确定性。本研究优化了档案材料定量逆转录-聚合酶链反应（RT-PCR）的RNA分离和反转录步骤。使用直接从手术室取出的组织，分离半衰期为10分钟至8小时的mrna并进行逆转录。随后对这些cdna的实时定量PCR方法（TaqMan）给出了固定组织中与新鲜组织中相当的基因表达测量。此外，我们模拟了常规病理处理，并证明这种mRNA定量方法对预固定时间（从切除到固定的时间）不敏感，最长可达12小时。因此，在组织收集程序很大程度上未知的存档组织中分析基因表达应该是可行的。
Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours, Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.