title: Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs.
abstract: microRNAs (miRNAs) hold promise as biomarkers for a variety of disease processes and for determining cell differentiation. These short RNA species are robust, survive harsh treatment and storage conditions and may be extracted from blood and tissue. Pre-analytical variables are critical confounders in the analysis of miRNAs: we elucidate these and identify best practices for minimizing sample variation in blood and tissue specimens. Pre-analytical variables addressed include patient-intrinsic variation, time and temperature from sample collection to storage or processing, processing methods, contamination by cells and blood components, RNA extraction method, normalization, and storage time/conditions. For circulating miRNAs, hemolysis and blood cell contamination significantly affect profiles; samples should be processed within 2 h of collection; ethylene diamine tetraacetic acid (EDTA) is preferred while heparin should be avoided; samples should be "double spun" or filtered; room temperature or 4 °C storage for up to 24 h is preferred; miRNAs are stable for at least 1 year at -20 °C or -80 °C. For tissue-based analysis, warm ischemic time should be <1 h; cold ischemic time (4 °C) <24 h; common fixative used for all specimens; formalin fix up to 72 h prior to processing; enrich for cells of interest; validate candidate biomarkers with in situ visualization. Most importantly, all specimen types should have standard and common workflows with careful documentation of relevant pre-analytical variables.
authors.affiliation: University of Washington, Department of Laboratory Medicine, Seattle, WA.
authors.forename: Jenna
authors.initials: J
authors.lastname: Khan
authors.affiliation: University of Washington, Department of Laboratory Medicine, Seattle, WA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Lieberman
authors.affiliation: University of Washington, Department of Laboratory Medicine, Seattle, WA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Lockwood
mesh_terms: D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards*; D001800:Blood Specimen Collection; D006801:Humans; D035683:MicroRNAs / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification; D012015:Reference Standards; D013997:Time Factors
chemical_list: D035683:MicroRNAs
keywords: FFPE; biomarker; circulating; miRNA; pre-analytical; standardization; tissue; variables