Popov Alexey, Szabo Arpad, Mandys Václav
Department of Pathology, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Srobarova 50, 100 00, Prague 10, Czech Republic.
BMC Cancer. 2015 Oct 24;15:774. doi: 10.1186/s12885-015-1785-9.
RT-qPCR quantification of miRNAs expression may play an essential role in pancreatic ductal adenocarcinoma (PDAC) diagnostics. RT-qPCR-based experiments require endogenous controls for the result normalization and reliability. However, expression instability of reference genes in tumors may introduce bias when determining miRNA levels.
We investigated expression of 6 miRNAs, isolated from FFPE samples of pancreatic adenocarcinomas. Four internal controls were utilized for RT-qPCR result normalization: artificial miR-39 from C. elegans, U6 snRNA, miR-16 and snoRNA U91.
We found miR-21, miR-155 or miR-217 expression values in tumors may differ up to several times, depending on selected internal controls. Moreover, different internal controls can produce controversial results for miR-96, miR-148a or miR-196a quantification. Also, expression of our endogenous controls varied significantly in tumors. U6 demonstrated variation from -1.03 to 8.12-fold, miR-16 from -2.94 up to 7.38-fold and the U91 from -3.05 to 4.36-fold respectively. On the other hand, the most stable gene, determined by NormFinder algorithm, was U91. Each miRNA normalized relatively to the spike or U91, demonstrated similar expression values. Thus, statistically significant and insignificant differences between tumors and normal tissues for miRNAs were equal for the spike and the U91. Also, the differences between the spike and U91 were statistically insignificant for all of miRs except miR-217. Among three endogenous controls, U91 had the lowest average expression values and standard deviation in cancer tissues.
We recommend U91 as a new normalizer for miRNA quantification in PDACs.
微小RNA（miRNA）表达的逆转录-定量聚合酶链反应（RT-qPCR）定量分析在胰腺导管腺癌（PDAC）诊断中可能发挥重要作用。基于RT-qPCR的实验需要内参基因来进行结果标准化和确保可靠性。然而，肿瘤中参考基因表达的不稳定性在确定miRNA水平时可能会引入偏差。
我们研究了从胰腺腺癌福尔马林固定石蜡包埋（FFPE）样本中分离出的6种miRNA的表达情况。使用了4种内参基因对RT-qPCR结果进行标准化：来自秀丽隐杆线虫的人工合成miR-39、U6小核RNA（snRNA）、miR-16和小核仁RNA U91。
我们发现，根据所选内参基因的不同，肿瘤中miR-21、miR-155或miR-217的表达值可能相差数倍。此外，不同的内参基因在miR-96、miR-148a或miR-196a定量分析中可能产生相互矛盾的结果。而且，我们的内参基因在肿瘤中的表达也存在显著差异。U6的变化范围为-1.03至8.12倍，miR-16为-2.94至7.38倍，U91为-3.05至4.36倍。另一方面，通过NormFinder算法确定的最稳定基因是U91。相对于加样对照或U91进行标准化后，每种miRNA都显示出相似的表达值。因此，对于miRNA，肿瘤组织与正常组织之间具有统计学意义和无统计学意义的差异，在加样对照和U91之间是相同的。此外，除miR-217外，加样对照与U91之间的差异对所有miR均无统计学意义。在三种内参基因中，U91在癌组织中的平均表达值和标准差最低。
我们推荐U91作为PDAC中miRNA定量分析的新的标准化内参基因。
