A quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples. Homo sapiens

MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p<0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). Our study aims to demonstrate the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies. Overall design: FFPE samples from 4 benign, reactive lymph nodes and 3 mantle cell lymphomas. Paired fresh-frozen and FFPE samples (3 benign reactive lymph nodes and 3 mantle cell lymphomas) were used for comparison. We also tested RNA samples at different concentrations (10, 25, 50, 100 and 200 ng/uL) as well as duplicate experiments for these RNA concentrations. Distinct RNA dilutions were also compared (5X, 7.5X, 30X and 62.5X dilution) at RNA concentrations of 66.7 ng/uL, 100 ng/uL and 200 ng/uL).

微小RNA（MicroRNAs，miRs）是一类参与转录后调控的非编码RNA分子，在组织发育、细胞分化、细胞增殖与细胞凋亡过程中发挥多种生物学功能。miRs在甲醛固定过程中不易降解，为福尔马林固定石蜡包埋（FFPE）组织的miRNA表达研究提供了便利。本研究证实，TaqMan人类微小RNA芯片v1.0（早期试用版）平台适用于FFPE组织的miRNA表达分析，且重复性极佳：重复样本间相关系数达0.95（p<0.001）。研究显示，技术重复、等倍稀释样本以及FFPE与新鲜冷冻样本之间的重复性在高丰度miRs层级中表现最优。本研究同时证实，无论采用何种RNA提取方法，在检测所有miRs时，FFPE样本与新鲜冷冻样本的miRNA表达谱具有可比性，相关系数最高可达0.87（p<0.001）。按miRs丰度分层分析FFPE与新鲜冷冻样本间的相关系数可见，低丰度miRs的相关系数最高可达0.32，中等丰度为0.70，高丰度则高达0.97。本研究旨在阐明基于高通量定量PCR的miRNA分析平台开展FFPE样本miRNA表达研究时所需的实用性、重复性及优化方案，为回顾性研究开辟广阔的研究空间。实验设计：本研究纳入4例良性反应性淋巴结及3例套细胞淋巴瘤的FFPE样本；同时采用3例良性反应性淋巴结与3例套细胞淋巴瘤的配对新鲜冷冻样本与FFPE样本进行对比分析。此外，我们测试了不同浓度（10、25、50、100及200 ng/μL）的RNA样本，并对这些浓度的RNA样本开展重复实验；在66.7 ng/μL、100 ng/μL及200 ng/μL的RNA浓度下，还对不同稀释梯度（5倍、7.5倍、30倍及62.5倍稀释）的RNA样本进行了比较。