Boisen Mogens Karsbøl, Dehlendorff Christian, Linnemann Dorte, Schultz Nicolai Aagaard, Jensen Benny Vittrup, Høgdall Estrid Vilma Solyom, Johansen Julia Sidenius
Department of Oncology, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730, Herlev, Denmark.
Statistics, Bioinformatics and Registry, Danish Cancer Society Research Center, Copenhagen, Denmark.
BMC Cancer. 2015 Dec 29;15:1024. doi: 10.1186/s12885-015-2030-2.
Archival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation.
High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering.
We created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50% reduction over 20 years) but not in CRC. Formalin fixation for 2-6 days decreased miRNA expression 30-65%. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results.
We identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE tissue samples. Formalin fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.
存档的福尔马林固定石蜡包埋（FFPE）癌症组织样本是用于鉴定微小RNA（miRNA）生物标志物的现成资源。目前尚无针对FFPE组织中参考miRNA的既定标准。我们试图鉴定稳定的参考miRNA，用于对结直肠癌（CRC）和胰腺癌（PC）患者的FFPE组织样本中的miRNA表达进行标准化，并量化与样本年龄和固定相关的变异性。
使用了来自203个CRC和256个PC FFPE样本以及来自其他9个CRC肿瘤的37对冷冻/FFPE样本（方法学样本）的高通量miRNA谱分析结果。通过与全局平均表达的相关性来鉴定候选参考miRNA。根据已发表的方法分析参考基因的稳定性。使用线性回归测试样本年龄与全局平均miRNA表达之间的关联。使用相关系数和线性混合效应模型描述变异性。通过标准差的变化和层次聚类确定标准化效果。
我们创建了在每种癌症类型中与全局平均表达相关性最佳的20个miRNA列表。其中9个miRNA同时出现在两个列表中，miR-103a-3p是CRC和PC FFPE组织中最稳定的参考miRNA。CRC中参考miRNA的最佳数量为4个，PC中为10个。样本年龄对PC中的全局miRNA表达有显著影响（20年内降低50%），但对CRC没有影响。福尔马林固定2 - 6天会使miRNA表达降低30 - 65%。使用全局平均表达进行标准化可降低技术和生物学重复的变异性，而使用鉴定出的参考miRNA的表达进行标准化仅可降低生物学重复的变异性。标准化对聚类结果的影响较小。
我们为未来使用CRC和PC FFPE组织样本进行的miRNA表达实验鉴定了合适的参考miRNA。福尔马林固定会显著降低miRNA表达，而在临床环境中，样本年龄增加的影响估计可忽略不计。
