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Coronary heart disease (CHD) is one of the leading causes of heart-associated deaths worldwide. Conventional diagnostic techniques are ineffective and insufficient to diagnose CHD with higher accuracy. To use the circulating microRNAs (miRNAs) as non-invasive, specific and sensitive biomarkers for diagnosing of CHD, 203 patients with CHD and 144 age-matched controls (126 high-risk controls and 18 healthy volunteers) were enrolled in this study. The direct S-Poly(T)Plus method was used to identify novel miRNAs expression profile of CHD patients and to evaluate their clinical diagnostic value. This method is an RNA extraction-free and robust quantification method, which simplifies procedures, reduces variations, in particular increases the accuracy. Twelve differentially expressed miRNAs between CHD patients and high-risk controls were selected, and their performances were evaluated in validation set-1 with 96 plasma samples. Finally, six (miR-15b-5p, miR-29c-3p, miR-199a-3p, miR-320e, miR-361-5p and miR-378b) of these 12 miRNAs were verified in validation set-2 with a sensitivity of 92.8% and a specificity of 89.5%, and the AUC was 0.971 (95% confidence interval, 0.948-0.993, P < .001) in a large cohort for CHD patients diagnosis. Plasma fractionation indicated that only a small amount of miRNAs were assembled into EVs. Direct S-Poly(T)Plus method could be used for disease diagnosis and 12 unique miRNAs could be used for diagnosis of CHD., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Advances in microRNAs (miRNAs) biomarkers have generated disease markers with potential clinical values. However, none of these published results have been applied in clinic until today. The main reason could be the lack of simple but robust miRNA measurements., Methods: We built up a simple but ultrasensitive RT-qPCR protocol, Direct S-Poly(T) Plus assay, for detecting miRNAs without RNA purification. In this study, the method was optimized and compared with other RNA purification-based miRNA assays, and the sensitivity was tested. Using Direct S-Poly(T) Plus method, seven potential miRNA biomarkers of colorectal cancer were validated., Results: It is possible to detect approximately 100 miRNAs with minimal plasma inputs (20 μl) and time (~ 140 min) with this approach. The sensitivity of this method was 2.7-343-fold higher than that of the stem-loop method, and comparable with S-Poly(T) plus method. 7 validated miRNA biomarkers of colorectal cancer by Direct S-Poly(T) plus assay could discriminate colorectal cancer stage I from healthy individuals, and promised satisfactory discrimination with the area under receiver operating characteristic (ROC) curve ranging from 0.79 to 0.94 (p value < 0.001)., Conclusions: This simple and robust protocol may have strong impact on the development of specific miRNAs as biomarkers in clinic.