‘Future-proofing’ blood processing for measurement of circulating microRNAs in samples from biobanks and prospective clinical trials14 Dec 2017
BACKGROUND: Quantifying circulating nucleic-acids is an important new approach to cancer diagnosis/monitoring. METHODS: We compared the suitability of serum versus plasma for measuring microRNAs using RT-qPCR and assessed how pre-analytical variables that can affect circulating-tumorDNA (ctDNA) quantification in plasma also influence microRNA levels. RESULTS: Across 62 blood-derived specimens, plasma samples in EDTA, Streck-DNA-plasma and Streck-RNA-plasma tubes showed significantly higher Ct values for multiple housekeeping microRNAs, compared with serum samples. For the EDTA-plasma tubes, this difference was only seen when including the high-speed centrifugation protocol used to optimise 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 microRNAs 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 microRNAs to those of hsa-miR- 451a, in order to obtain the stable values needed for referencing test microRNA levels. CONCLUSIONS: Our data indicate that plasma samples prepared for ctDNA extraction are suboptimal for microRNA quantification and require the incorporation of multiple data normalization steps. For prospective studies designed to measure both microRNAs and ctDNA, the most suitable approach would be to obtain both serum (for microRNAs) 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 microRNA quantification, and the other for a further high-speed centrifugation step to optimize ctDNA retrieval. IMPACT: These recommendations will help ‘future-proof’ clinical studies in which quantification of circulating microRNAs is a component.