Establishment of preanalytical conditions for microRNA profile analysis of clinical plasma samples

Publication Date: 14 December 2022

Suzuki, K. et al. (2022) Establishment of preanalytical conditions for microRNA profile analysis of clinical plasma samples. PLoS ONE 17, e0278927. DOI: 10.1371/journal.pone.0278927

Previous studies have shown that individuals with and without cancer have different microRNA (miRNA) expression levels. MiRNAs are 20–25 nucleotide noncoding RNAs that regulate gene expression by inducing mRNA degradation and inhibiting mRNA transcription. Early cancer detection and treatment improves survival rates but screening for multiple cancers require multiple samples from patients. A blood-based screening method would reduce the screening burden on patients. Technologies like next-generation sequencing (NGS) make it easy to examine the expression profiles of over 2,500 miRNAs at once. However, previous miRNA cancer association studies produced inconsistent results that could be due to variations in blood handling and storage.

This study examined the effects of anticoagulants and storage conditions on mRNA expression to standardize a protocol that could better provide reproducible NGS profiling data. Blood samples were gathered from healthy individuals to evaluate collection conditions and find the best parameters for reproducible miRNA results. Plasma samples were prepared and RNA extracted using automated methods before preparing cDNA libraries for miRNA expression analysis. Library concentration, identification rate, the unique molecular identifier (UMI) ratio and expression levels of erythrocyte- and platelet-derived miRNAs were evaluated. The researchers determined collecting blood in EDTA tubes had the highest RNA concentration and exhibited the lowest lysis compared to sodium fluoride and sodium citrate tubes. Storage time and temperature of both whole blood and plasma were tested to determine the best storage and handling conditions. The conditions tested were blood stored at room temperature from 1 hours to 3 days, plasma stored at 4°C and –20°C from 1 hour to 30 days and plasma banked at –80°C over 3, 4 and 5 years. During the study, several miRNAs were identified as time-dependent markers that increased or decreased over time in whole blood.

Based on the results of blood and plasma samples, researchers applied what they learned to blood samples from cancer patients. Three facilities were asked to collect 20 blood samples using EDTA blood collection tubes, refrigerate within 1 hour, separate plasma within 12 hours, and store the plasma samples at –80°C. There were no significant differences in the 60 samples collected from the facilities based on library concentration, identification rate and UMI ratio. However, there was some variation in hemolysis among the facilities, but did not exceed the variation observed from samples drawn at the same facility. Researchers concluded that a reproducible miRNA expression profile was possible based on the blood handling and storage conditions they tested during their study.

Keywords: Specimen storage, blood plasma, blood, cancers and neoplasms, microRNA profiling, NGS, health care facilities