Abstract 4583: Biospecimen banking research with circulating microRNAs: internal controls and pre-analytic variables.
Authors: Zhao Hua , Liu Song , Hu Qiang , Yan Li , Davis Warren , Nesline Mary , Ambrosone Christine 
Published: August 2014

Abstract
Because of the significance of microRNA (miRNA) in carcinogenesis, circulating miRNAs offer unique opportunities as minimal invasive biomarkers for cancer diagnosis and prognosis. To date, more than 200 studies have been reported. However, no single miRNA has been consistently observed across the studies. The wide inconsistency might be due to the fact that a significant amount of pre-analytic variations occurs from blood collection to the molecular analysis, and these variations may significantly contribute to observed inter-individual and intra-individual variations in circulating miRNAs. Little is known about how pre-analytic variations might affect the levels of circulating miRNAs, and there are no reliable internal controls currently available. Therefore, using high-quality biospecimens collected from the DataBank and BioRepository (DBBR) of Roswell Park Cancer Institute (RPCI), we performed a study to identify circulating miRNAs which can be used internal controls and then use them to study the effect of selected pre-analytic variables on circulating miRNAs in EDTA-plasma and whole blood collected using PaxGene tubes. Our criteria for internal control miRNAs include: they occur in all tested samples; their expression levels are not significantly different between cancer cases and controls; they show little inter-individual variations among cases and controls. Using exiqon miRNA PCR panel analysis, we performed miRNA profiling in 40 cancer cases and 40 healthy controls. A total of 4 miRNAs in whole blood (miR-134, miR-346, miR-934 and miR-16) and one miRNA in plasma (miR-16) fit our selection criteria for internal control miRNAs. These candidates were further validated in additional 100 cancer cases and 100 healthy controls. Using these identified internal control miRNAs, we next studied the effect of selected pre-analytic variables, including processing delay time, storage conditions, storage durations, and cycles of freeze/thaw. Twenty-four hour processing delay did not affect three tested miRNAs in whole blood, but significantly decreased the levels of miR-16 in plasma (P&lt;0.01). Increasing cycles of freeze/thaw from 0 to 1, 2 in whole blood, and 0 to 1, 2, 4 in plasma significantly decreased the levels of tested circulating miRNAs in both whole blood and plasma. Inverse dose response relationships were observed (P&lt;0.01 for both whole blood and plasma). Storage condition variations (Plasma: cryovials at -80°C vs straws in liquid nitrogen; Whole blood: -20°C vs -80°C) and storage duration variations (0 vs 6 months) seemed to have no effect on levels of tested circulating miRNAs in both whole blood and plasma. Our data clearly demonstrate the impact of pre-analytic variables on circulating miRNAs. Further biospecimen research is urgently needed to optimize the potential use of circulating miRNAs in cancer management. Funded by NCI Contract No. HHSN261200800001E
Citation Format: Hua Zhao, Song Liu, Qiang Hu, Li Yan, Warren Davis, Mary Nesline, Christine Ambrosone. Biospecimen banking research with circulating microRNAs: internal controls and pre-analytic variables. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4583. doi:10.1158/1538-7445.AM2013-4583