International Journal of Molecular Medical Science, 2025, Vol.15, No.4, 175-184 http://medscipublisher.com/index.php/ijmms 178 blotting (WB) was used to detect surface markers of exosomes (such as CD63, CD9, HSP70) and to check for impurities (Malla et al., 2018; Sanz-Rubio et al., 2018). The combined use of these methods can ensure the reliability of subsequent miRNA analysis results. 4.2 Selection of high-throughput miRNA detection platform and data quality control Common methods for simultaneously detecting a large number of mirnas include sequencing (NGS), microarray or quantitative PCR (RT-qPCR). NGS can measure more types and has high sensitivity. RT-qPCR is more accurate, has a wider measurement range, and is more suitable for verifying known mirnas (Figure 1) (Cheng et al., 2014; Ding et al., 2018; Grunt et al., 2020; Rahimian et al., 2023). Which method to choose depends on what research you want to conduct, the number of samples, and whether you want to find new mirnas or confirm the known ones. Figure 1 Amplification of miRNAs isolated from different sample types and volumes (Adopted from Grunt et al., 2020) Image caption: MiRNAs were extracted from MGP-derived exosomes of increasing plasma (EDTA) and serum volumes by our newly established miRNA isolation method and amplified by our modified RT-q PCR. The Cq values derived from the real-time PCR are shown in the tables for miR-16 (A) and miR-142 (B). In addition, the Cq values of exosomal (MGP-isolated pellet) and cell-free (supernatant) miR-16 and miR-142 derived from 500 µl of plasma (EDTA and CPD) and serum are compared (C) (Adopted from Grunt et al., 2020) Each step requires strict data quality control. This includes using controls to monitor RNA extraction efficiency, examine RNA integrity, and evaluate the differences between experiments of the same batch and different batches (Occhipinti et al., 2016; Malla et al., 2018). Sample processing, preservation and operation must be consistent, as
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