International Journal of Molecular Medical Science, 2025, Vol.15, No.4, 175-184 http://medscipublisher.com/index.php/ijmms 179 repeated freeze-thaw or hemolysis can affect the stability and measurement results of exosomal miRNA (Cheng et al., 2014; Sanz-Rubio et al., 2018). These quality control measures ensure the repeatability and reliability of miRNA data. 4.3 Data standardization and batch effect correction The standardization of miRNA data is crucial as it can reduce technical differences and enable accurate comparisons among different samples. For RT-qPCR data, it is recommended to select stable internal reference genes (such as let-7a or artificially added control RNA), and tools such as BestKeeper and NormFinder can be used to help find the best internal reference (Occhipinti et al., 2016; Sanz-Rubio et al., 2018). For NGS or chip data, quantile normalization or normalization methods based on the overall expression level are commonly used (Rahimian et al., 2023). The processing of samples at different time points, the use of reagents with different batch numbers, or the operation of instruments (referred to as batch effect) can affect the results. Statistical methods (such as batch effect correction algorithms) or increasing repeated experiments can be used to reduce this influence (Occhipinti et al., 2016; Rahimian et al., 2023). Correct standardization and batch correction are of great significance as they ensure that the detected miRNA differences truly reflect biological changes rather than experimental errors. 5 The Differences in miRNA Characteristics of Exosomes in the Blood of Patients with Alzheimer's Disease 5.1 The overall pattern of elevated and decreased miRNA Analysis of exosomal mirnas in the blood of patients with Alzheimer's disease (AD) shows that the expression levels of many mirnas have changed compared with those of healthy people, with some increasing and some decreasing. For instance, through sequencing and qRT-PCR verification, dozens of mirnas with significantly altered expression levels were identified - one study found 24 differentially expressed mirnas (with changes of more than 2 times), and another study found as many as 140, among which 82 were elevated in AD and 58 were decreased (Leidinger et al., 2013; Dong et al., 2021). These overall changes indicate that AD is associated with extensive alterations in exosomal mirnas, reflecting the underlying mechanisms of the disease. These altered mirnas are involved in a variety of biological activities, including cell connection, gene regulation, protein degradation systems, and processes related to neurodegeneration. It is particularly worth noting that in AD patients, miR-342-3p, miR-125b and miR-193b are often found to be decreased, while miR-30b-5p, miR-22-3p and miR-378a-3p are often found to be elevated, suggesting that they may be reliable biomarkers (Leidinger et al., 2013; Lugli et al., 2015; Yang et al., 2018; Manna et al., 2020; Dong et al., 2021; Duan et al., 2024). These patterns can be observed in various studies and detection methods, indicating that the results are reliable and clinically relevant. 5.2 Specific miRNA characteristics at different stages of the disease At different stages of AD, such as mild memory problems (MCI), early AD and late dementia, the characteristics of exosomal mirnas in the blood also vary. For example, mirnas such as miR-193b and miR-342-3p gradually decrease from MCI to the dementia stage; Mirnas like miR-30b-5p and miR-378a-3p have already changed in the early stage of the disease (Yang et al., 2018; Manna et al., 2020; Dong et al., 2021). This stage specificity makes them more valuable in early detection and disease tracking. Furthermore, combinations composed of multiple mirnas (such as 12 or 16 mirnas) are highly accurate in differentiating AD from healthy individuals or other brain diseases. The recognition ability (sensitivity and specificity) of some combinations exceeds 90% (Leidinger et al., 2013; Cheng et al., 2014; Hill et al., 2014). These characteristics not only reflect whether a disease exists or not, but are also related to the severity of the disease, supporting their use to track the progression of the disease and guide possible treatments. 5.3 Association between miRNA levels, cognitive scores and brain scan Indicators Multiple studies have shown that the level of exosomal miRNA in the blood is significantly associated with the
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