Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103/E, B-1090 Brussels, Belgium.
Department of Radiation Oncology, London Regional Cancer Program, London Health Sciences Centre, 800 Commisioners Road East, London, ON N6A 4G5, Canada.
Biomolecules. 2021 Apr 22;11(5):621. doi: 10.3390/biom11050621.
The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA's compatibility with the nanoString nCounter technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter technology.
基因表达谱（GEP）在癌症管理中的应用正在增加，因为 GEP 可用于疾病分类和诊断，根据药物反应的潜在遗传决定因素调整治疗方法，监测治疗反应和预后。然而，GEP 的可靠性在很大程度上取决于输入的 RNA 的数量和质量。这突出表明需要标准程序来确保从经常进行小肿瘤活检的组织中提取 RNA 时采用最佳实践，这些活检的组织处理存在差异。我们优化了一种从乳腺癌患者的新鲜冷冻（FF）芯针活检（CNB）和福尔马林固定石蜡包埋（FFPE）组织中提取 RNA 的方案，当 FF CNB 未产生足够的 RNA 时，使用该方案。研究了避免核糖核酸酶、组织匀浆或脱蜡以及组织成分对 RNA 提取的影响的方法。此外，还研究了 RNA 与 nanoString nCounter 技术的兼容性。该技术平台可使用小 RNA 片段进行 GEP。在优化方案后，92%的样本中从 FF CNB 中获得了高质量和足够数量的 RNA。对于其余 8%的情况，使用病理科制备的 FFPE 材料进行 RNA 提取。两种 RNA 终产物都与 nanoString nCounter 技术兼容。
