Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy

Abstract

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross‐studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin® kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter‐sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT‐qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two‐fold lower inter‐sample variability than the widely used endogenous miR‐16‐5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up‐to 0.97 between the microarrays and individual RT‐qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice.

Challenges in Using Circulating Micro-RNAs as Biomarkers for Cardiovascular Diseases

“…Though, this finding needs to be confirmed in different laboratories with a larger and diverse number of samples and different classes of miRNAs. In a most recent study, Vigneron et al [120] compared three commercially available isolation kits, i.e., miRNeasy Serum/Plasma, mirVana, and NucleoSpin miRNA Plasma, which had recently been made available. In this study, higher concentrations of miR-16-5p were obtained by the NucleoSpin kit compared to miRNeasy and mirVana kits.…”

Altered microRNA expression during Impaired Glucose Tolerance and High-fat Diet Feeding

“…Data normalization poses a significant challenge during miRNA profiling, particularly circulating miRNAs. Although there is no consensus about the best quantification method to use when profiling extracellular miRNAs, strategies based on exogenous miR-NAs such as Caenorhabiditis elegans miR-39 have been shown to be less variable than using endogenous miRNAs [32].…”

Exosomes and diabetes

“…Isolation by ultracentrifugation, precipitation, ultrafiltration, and other methodologies have been variously used, resulting in different EV populations, and making it difficult to compare studies . Finally, a method to normalize the data to the sample volume used is still needed, particularly in the case of miRNAs, whose quantification frequently relies on relative methods such as real‐time RT‐PCR …”