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

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, p<0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (3035), 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. 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、细胞增殖与凋亡过程中发挥多样功能。微小RNA在福尔马林固定过程中不易降解，这为福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）组织的微小RNA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）平台适用于福尔马林固定石蜡包埋组织的微小RNA表达分析，且具有极高的重复性（重复样本间相关系数达0.95，p<0.00001）；同时基于临床福尔马林固定石蜡包埋样本，明确了该平台的最优运行条件。我们提出了一种数据分析方法，用于对比福尔马林固定石蜡包埋样本与新鲜冷冻样本的微小RNA丰度差异：将检测到的微小RNA按丰度分为高丰度（Ct值<30）、中丰度（30< Ct <35）与低丰度（Ct>35）三个层级。研究结果显示，技术重复样本、等比例稀释样本以及福尔马林固定石蜡包埋与新鲜冷冻样本间的重复性，在高丰度层级中表现最优。此外，无论采用何种RNA提取方法，当分析所有微小RNA时，福尔马林固定石蜡包埋样本与新鲜冷冻样本的微小RNA表达谱具有良好的一致性，最高相关系数可达0.87（p<0.001）。按微小RNA丰度层级对比福尔马林固定石蜡包埋与新鲜冷冻样本的相关系数时可见，低丰度层级相关系数最高达0.32，中丰度层级达0.70，高丰度层级则可达0.97。本研究旨在验证基于高通量定量PCR的微小RNA检测平台，在福尔马林固定石蜡包埋样本的微小RNA表达研究中的实用性、重复性与优化方案，为回顾性研究拓展了广阔的研究空间。本研究使用的福尔马林固定石蜡包埋样本包括4例良性反应性淋巴结样本与3例套细胞淋巴瘤样本。研究同时选取配对的新鲜冷冻与福尔马林固定石蜡包埋样本（3例良性反应性淋巴结样本与3例套细胞淋巴瘤样本）用于组间对比。此外，我们还测试了不同浓度（10、25、50、100及200 ng/μL）的RNA样本，并针对上述浓度的RNA样本开展了重复实验。我们还在66.7 ng/μL、100 ng/μL及200 ng/μL的RNA浓度下，对比了不同稀释倍数（5倍、7.5倍、30倍及62.5倍）的RNA样本。