Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio Av. 7, 10257, Vilnius, Lithuania.
Institute of Endocrinology, Medical Academy, Lithuanian University of Health Sciences, Eiveniu Str. 2, 50161, Kaunas, Lithuania.
Mol Med. 2020 Nov 5;26(1):100. doi: 10.1186/s10020-020-00218-7.
The most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression.
RQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years.
The stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time.
It is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.
最流行的 miRNA 定量技术是 RQ-PCR 相对基因表达法，该方法需要内参（EC）基因进行数据归一化。然而，在许多癌症类型包括甲状腺乳头状癌（PTC）中，miRNA 表达研究中最适合的 EC 数据不足，选择标准也不明确。因此，在这项研究中，我们评估了所选 EC 和存档福尔马林固定、石蜡包埋（FFPE）PTC 组织年龄对估计的靶 miRNA 表达的影响。
使用 RQ-PCR 测定 5 种 miRNA（miR-146b、miR-222、miR-21、miR-221 和 miR-181b）和 3 种不同内参（RNU48、let-7a、miR-16）的表达水平，以归一化数据。总共分析了 400 个 FFPE PTC 组织，这些组织的储存时间从 1 年到 15 年不等。
常用 ECs RNU48 和 let-7a 的稳定性与 FFPE PTC 组织中靶 miRNA 的稳定性显著不同。此外，当 FFPE 组织样本储存时间不同时，这些差异对 miRNA 表达结果有很大影响。
选择不仅在感兴趣的组织中稳定，而且与靶 miRNA 具有相似稳定性的 ECs 非常重要，特别是在处理不同年龄的样本时。
