Chen Jun, Byrne Gerald E, Lossos Izidore S
Department of Medicine, Division of Hematology-Oncology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA [corrected]
Diagn Mol Pathol. 2007 Jun;16(2):61-72. doi: 10.1097/PDM.0b013e31802f0804.
Comprehensive analysis of gene expression using RNA extracted from frozen lymphoma specimens is becoming increasingly important for understanding disease pathogenesis, disease subclassification, and prognostication. As paraffin tissues are widely available whereas frozen specimens are not, development of gene expression analysis based on RNA extracted from paraffin-embedded tissues would facilitate application of the accumulated knowledge to a sample type that is typical of clinical practice. In the present study, we have developed and optimized methods of RNA extraction from paraffin-embedded lymphoid tissues. In contrast to previously suggested methods of RNA extraction from paraffin, our method uses sodium dodecyl sulfate that better preserves the extracted RNA and is optimized for more complete proteinase K digestion to release RNA from its complexes with protein. These modifications yield long RNA fragments up to 2000 bp enabling amplification of long amplicons. This allows usage of paraffin specimens for molecular rescue of RNA transcripted from rearranged clonal immunoglobulin genes-an advance that may increase the eligibility of lymphoma patients for immunotherapeutic approaches. Furthermore, real-time polymerase chain reaction analysis of expression of genes implicated in determination of prognosis of diffuse large B-cell lymphoma patients demonstrated an extremely high correlation (R>0.90) in normalized gene expression between paired frozen and formalin-fixed, paraffin-embedded specimens. Similarly, good correlation was also observed in gene array studies. These results suggest that the methods of RNA extraction we propose are suitable for giving accurate real-time quantitative reverse transcriptase-polymerase chain reaction results, array gene expression profiling, and molecular rescue of RNA transcripted from rearranged immunoglobulin genes for diagnostic and immunotherapeutic approaches.
使用从冷冻淋巴瘤标本中提取的RNA进行基因表达的综合分析，对于理解疾病发病机制、疾病亚分类和预后判断变得越来越重要。由于石蜡组织广泛可得而冷冻标本并非如此，基于从石蜡包埋组织中提取的RNA开展基因表达分析，将有助于把积累的知识应用于临床实践中典型的样本类型。在本研究中，我们开发并优化了从石蜡包埋淋巴组织中提取RNA的方法。与先前提出的从石蜡中提取RNA的方法不同，我们的方法使用十二烷基硫酸钠，它能更好地保存提取的RNA，并且经过优化以实现更完全的蛋白酶K消化，从而从与蛋白质的复合物中释放RNA。这些改进产生了长达2000 bp的长RNA片段，能够扩增长扩增子。这使得石蜡标本可用于对重排的克隆免疫球蛋白基因转录的RNA进行分子挽救——这一进展可能会增加淋巴瘤患者接受免疫治疗的可能性。此外，对弥漫性大B细胞淋巴瘤患者预后相关基因表达的实时聚合酶链反应分析表明，配对的冷冻标本与福尔马林固定石蜡包埋标本之间的标准化基因表达具有极高的相关性（R>0.90）。同样，在基因阵列研究中也观察到了良好的相关性。这些结果表明，我们提出的RNA提取方法适用于给出准确的实时定量逆转录聚合酶链反应结果、阵列基因表达谱分析，以及对重排免疫球蛋白基因转录的RNA进行分子挽救以用于诊断和免疫治疗方法。
