Bioinformatics Core, Department of Complementary & Integrative Medicine , University of Hawaii John A. Burns School of Medicine , Honolulu , Hawaii 96813 , United States.
National Center of Colorectal Disease, Nanjing Municipal Hospital of Chinese Medicine, the Third Affiliated Hospital , Nanjing University of Chinese Medicine , Nanjing 210001 , P. R. China.
Anal Chem. 2019 May 21;91(10):6746-6753. doi: 10.1021/acs.analchem.9b00821. Epub 2019 Apr 30.
Recent studies have indicated that circulating noncoding RNAs (ncRNAs) such as miRNAs are stable biomarkers for the diagnosis and prognosis of human diseases. However, due to low concentrations of circulating ncRNAs in blood, data normalization in plasma/serum ncRNA experiments using next-generation sequencing and quantitative real time RT-qPCR is a challenge. We found that the current normalization methods based on synthetic external spiked-in controls or published endogenous miRNA controls are inappropriate as they are not stably expressed and therefore fail to reliably detect differentially expressed ncRNAs. Using the alternative of individual ncRNAs as biomarkers, we considered a ratio-based normalization method calculated taking the ratio of any two ncRNAs in the same sample and used the resulting ratios as biomarkers. We mathematically verified the method to be independent of spiked-in and internal controls, and more robust than existing reference control based normalization methods to identify differentially expressed ncRNAs as potential biomarkers for human diseases. Thus, the ratio-based method can solve the difficult normalization problem for circuiting ncRNA data to identify reliable biomarkers to meet real clinical practice.
最近的研究表明，循环非编码 RNA（ncRNA），如 miRNAs，是人类疾病诊断和预后的稳定生物标志物。然而，由于血液中循环 ncRNA 的浓度较低，使用下一代测序和定量实时 RT-qPCR 对血浆/血清 ncRNA 实验进行数据标准化是一个挑战。我们发现，目前基于合成外源性添加对照或已发表的内源性 miRNA 对照的标准化方法是不适当的，因为它们的表达不稳定，因此无法可靠地检测差异表达的 ncRNA。使用个体 ncRNA 作为生物标志物的替代方法，我们考虑了一种基于比率的标准化方法，该方法通过计算同一样本中任何两个 ncRNA 的比率，并将得到的比率用作生物标志物。我们从数学上验证了该方法不受添加物和内参的影响，并且比现有的基于参考控制的标准化方法更稳健，可以识别差异表达的 ncRNA 作为人类疾病的潜在生物标志物。因此，基于比率的方法可以解决循环 ncRNA 数据的标准化难题，以确定可靠的生物标志物，以满足实际的临床实践。
