Evaluation and validation of total RNA extraction methods for MicroRNA expression analyses in formalin-fixed, paraffin-embedded tissues
Evaluation and validation of total RNA extraction methods for MicroRNA expression analyses in formalin-fixed, paraffin-embedded tissues
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DOI：
10.2353/jmoldx.2008.070153
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发表时间：
2008-05-01
影响因子：
4.1
通讯作者：
Szafranska, Anna E.
中科院分区：
文献类型：
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作者：
Doleshal, Martina;Magotra, Amber A.;Szafranska, Anna E.
福尔马林固定、石蜡包埋（FFPE）组织标本的组织学档案是人类疾病回顾性研究的宝贵资源。由于质量完整的mRNA兼容的分子技术的恢复往往是困难的，由于降解，我们评估了microRNA（miRNA），一类新的小RNA分子与日益增长的治疗和诊断潜力，作为一种替代分析物的FFPE样品的基因表达研究。通过分析总RNA产量、miRNA回收率和实时聚合酶链反应对miRNA、mRNA和rRNA种类的稳健性，我们比较了市售RNA分离试剂盒的性能，并确定了优选的方法。我们进一步实施了修改，以增加组织通量，并纳入定量装甲RNA过程控制，以监测RNA回收效率。对优化的工艺进行了重现性、操作员和日间变异性测试，并使用一组30份临床样本进行了验证。此外，我们证明，独立于FFPE块的年龄和RNA质量，miRNA产生的定量逆转录-聚合酶链反应信号更强大，更好地与冷冻参考样本的表达水平相关，与较长的mRNA相比。我们的广泛研究，包括来自17种组织类型的总共272个独立的RNA分离物和长达12年的65个FFPE块，表明miRNA不仅适合，而且可能是受损存档临床标本的分子表征的上级分析物。
Histopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens.