“Future-Proofing” Blood Processing for Measurement of Circulating miRNAs in Samples from Biobanks and Prospective Clinical Trials

Abstract

Quantifying circulating nucleic acids is an important new approach to cancer diagnosis/monitoring. We compared the suitability of serum versus plasma for measuring miRNAs using qRT-PCR and assessed how preanalytic variables that can affect circulating tumor DNA (ctDNA) quantification in plasma also influence miRNA levels. Across 62 blood-derived specimens, plasma samples in EDTA, Streck-DNA, and Streck-RNA tubes showed significantly higher values for multiple housekeeping miRNAs, compared with serum samples. For the EDTA-plasma tubes, this difference was only seen when including the high-speed centrifugation protocol used to optimize ctDNA extraction. In plasma samples derived from blood stored at room temperature for up to 14 days (conditions that typically apply to samples processed for biobanking), levels of endogenous housekeeping miRNAs gradually increased, in parallel with the hemolysis marker hsa-miR-451a, consistent with release from blood cells/platelets. It was necessary to normalize levels of the housekeeping miRNAs to those of hsa-miR-451a, to obtain the stable values needed for referencing test miRNA levels. Our data indicate that plasma samples prepared for ctDNA extraction are suboptimal for miRNA quantification and require the incorporation of multiple data normalization steps. For prospective studies designed to measure both miRNAs and ctDNA, the most suitable approach would be to obtain both serum (for miRNAs) and plasma (for ctDNA). If only plasma can be collected, we recommend an initial low-speed centrifugation step, followed by aliquoting the supernatant into parallel samples, one for direct miRNA quantification, and the other for a further high-speed centrifugation step to optimize ctDNA retrieval. These recommendations will help "future-proof" clinical studies in which quantification of circulating miRNAs is a component. .

Serum Levels of MicroRNA-371a-3p (M371 Test) as a New Biomarker of Testicular Germ Cell Tumors: Results of a Prospective Multicentric Study

“…All serum samples were deep frozen for several months until measurement; thus, some sample deterioration may have occurred in the meantime. 42 Strengths of the study involve the multicentric, multinational design, which resulted in a minimal selection bias. In addition, laboratory measurement techniques were uniformly used for all samples analyzed.…”

Serum Levels of miR-148a and miR-21-5p Are Increased in Type 1 Diabetic Patients and Correlated with Markers of Bone Strength and Metabolism

“…Data analysis was performed using 2 −Δ C t method; samples with resulting raw cycle-threshold ( C t ) >35.0 were considered as not detected/not expressed. Normalization analysis was performed by using miRNAs hsa-miR-451 to exclude hemolyzed samples alongside with 414 nm spectrophotometer absorbance [70,71,72], hsa-miR-191 as endogenous control previously reported by other studies [73,74,75] and exogenous ath-miR-159a as technical control of RNA extraction reproducibility [76,77].…”

Prognostic significance of circulating microRNA-214 and -126 in dogs with appendicular osteosarcoma receiving amputation and chemotherapy

“…Currently, there is no validated method for correcting miRNA levels affected by hemolysis in dogs. However, several markers are proposed for calibration of miRNAs in human hemolytic blood [49, 50]. The combination use of these hemolysis markers could further improve the accuracy of the prognostic prediction using these miRNAs after the validation in dogs.…”