Influence of pre-analytical conditions on cell-free microRNA stability in blood plasma samples

In a study published today in the ExRNA journal, researchers from the Novosibirsk Institute of Chemical Biology and Fundamental Medicine have proved that pre-analytical variables, as blood plasma or extracellular vesicles storage conditions influence on miRNAs stability and, thus, detected concentrations of individual microRNAs.

Many scientists around the world are trying to improve the quality of diagnostics or prognostic of diseases, including oncological ones, using various body fluids (blood, urine, saliva, etc.). MicroRNA molecules (18 to 25 nucleotides in size) are well known posttranscriptional regulators of gene expression and intracellular communication molecules. Human biological fluids contain about 3,000 individual microRNA, releasing from normal or tumor cells. MicroRNA circulate in biological fluids in different complexes with biopolymers and being packed in microvesicles, are rather stable and thus are considered as a convenient source of diagnostic material for liquid biopsy of tumors or other pathological states.

To be used as biomarkers, universally accepted systematic guidelines of bio samples processing based on knowledge, including storage stability of microRNA, must be elaborated.

“In our laboratory, work is being carried out to study cell-free microRNAs in the blood and urine during the development of various oncological diseases,” - says Ivan Zaporozhchenko, PhD, one of the main contributors to this study, - “at the beginning of our research, we were faced with the choice of not only the best source of diagnostic/prognostic microRNAs (simply in combination with blood/urine proteins or as part of extracellular vesicles of these biological fluids), but also a method and conditions for storing of microRNA containing samples”.

“Pre-analytical conditions yielding reproducible results should be explored in detail and properly validated in order to establish a gold standard protocol for sampling and processing of biological fluids for analysis of microRNA expression,” says Ivan Zaporozhchenko.

This study aimed to evaluate the influence of plasma storage conditions on stability of endogenous miRNAs in human blood plasma. We report stability of four endogenous miRNAs (-16, -19b, -23a, -451a) and cel-miR-39 as exogenous miRNA under short and long-term incubation at different temperatures as well as the effect of long-term storage on EVs (extracellular vesicles) miRNAs stability. Additionally, we have compared miRNA yields from fresh and archived blood plasma samples and effects of variations in miRNA extraction protocol and RNA protective agent on isolation efficiency. Important disclaimer is that in this study we use single-phase miRNA isolation method, which we have successfully used in several studies aimed at searching miRNA biomarkers of lung cancer in blood.

In our study, we confirm that the degradation rate of miRNAs can be affected by their structure and packaging. Addition of various stabilization solutions to biofluids can affect the efficiency of miRNA extraction.

Overall, the results of our long-term studies demonstrate the need to analyze cell-free nucleic acids, including microRNA, within 2-4 weeks after receiving biological samples.

Martínez Pabón B, Zaporozhchenko I, Konoshenko M, Murina E, Bryzgunova O, et al. Influence of pre-analytical conditions on cell-free microRNA stability in blood plasma samples. ExRNA 2025(1):0005, https://doi.org/10.55092/exrna20250005.