Detection and quantification of extracellular microRNAs in murine biofluids
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Biological Procedures Online
METHODOLOGY
Open Access
Detection and quantification of extracellular microRNAs in murine biofluids Thomas C Roberts1,2*, Anna M L Coenen-Stass1, Corinne A Betts1 and Matthew J A Wood1
Abstract Background: MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest. Results: The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used. Conclusions: Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity. Keywords: Extracellular microRNA, miRNA, Biofluid, RT-qPCR, Serum, Plasma
Background MicroRNAs (miRNAs) are short RNA molecules involved in gene regulation in higher organisms [1] which have recently been detected in extracellular biofluids including serum/plasma [2,3], urine [4], cerebral spinal fluid [5], saliva [6] and seminal fluid [7]. Since their discovery in 1993 [8] miRNAs have been the subject of intense study on account of their involvement in a plethora of physiological and pathophysiological processes. The presence of miRNAs in biofluids was somewhat surprising, as the extracellular space is an RNase-rich environment where synthetic RNA oligonucleotides are rapidly degraded (within seconds) after systemic injection [9]. It is now known that miRNAs are protected from nucleolytic degradation by encapsulation within extracellular vesicles [10-12], or * Correspondence: [email protected] 1 Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, OX1 3QX Oxford, UK 2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, 92037 La Jolla, CA, USA
by forming complexes with proteins and/or lipoproteins [13-15]. Given that miRNAs are abundant and highly stable in biofluids they have attracted much
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