Читать книгу Genomic and Epigenomic Biomarkers of Toxicology and Disease - Группа авторов - Страница 64

Although using circulating miRNA as biomarkers has its advantages, since this is a relatively non-invasive technique and also one that can in principle be used in the early diagnosis of diseases, miRNA profiling in biofluids is still in its infancy. Therefore it is necessary to highlight limitations that may lead to inconsistent findings. The identification of potential confounding factors will also help optimize the reproducibility of miRNA future biomarker studies used in metal toxicology.

Perhaps the biggest challenge to the successful identification of circulating biomarkers lies in the technical constraints on isolating and purifying the samples for analysis. Methods used by different groups to isolate miRNAs from blood vary greatly, which may contribute to reproducibility issues (Mariner et al. 2018). For instance, previous studies have shown that the differential expression analysis of circulating miRNA from plasma can be confounded by the presence of platelets, which require additional steps of centrifugation in order to accurately quantify differential circulating miRNA expression profiles (Cheng et al. 2013). However, one protocol to address this issue has already been developed (Mariner et al. 2018). Additionally, the type of RNA extraction protocol can vary from lab to lab. Traditional approaches to extracting RNA use phenol:chloroform steps to separate RNAs from proteins, then precipitation of RNA with alcohol and pelleting by centrifugation (Mariner et al. 2018). But the size and quantity of circulating miRNAs may make them difficult to pellet, and this leads to greater sample variability. Furthermore, commercialized kits that use column-based purification techniques such as RT-qPCR, next-generation sequencing, or microarray approaches may inhibit the most common downstream miRNA quantification as a result of the incomplete removal of denaturants.

Other challenges—for instance the normalization of data to a housekeeping circulating miRNA or other genes, low concentration of circulating miRNAs, and identification of acceptable ranges for certain circulating miRNAs in profiles of normal individuals—can contribute to data variability or impede meaningful interpretations (Cui et al. 2019). One study has addressed the effect of different normalization strategies to quantify the circulating miRNAs in human plasma and has found that miR-320d may be the most reliable endogenous circulating miRNA to use for normalization (Faraldi et al. 2019). Alternatively, others have used exogenous, synthetic miRNA mimic from C. elegans as normalization controls (Farina et al. 2014). Although according to some studies storage conditions do not seem to play a role in affecting the stability of circulating miRNAs, multiple freeze thaw cycles should be avoided in order to reduce the degradation of limited circulating miRNA species, and samples should kept at -80 C for long-term storage (Farina et al. 2014; Tiberio et al. 2015). Lastly, no studies to date have addressed the matter of a normal physiological range for circulating miRNAs. However, on the basis of individual variability in circulating miRNAs, these studies are needed before the use of circulating miRNAs as biomarkers in a clinical setting.