title: Detection and quantification of extracellular microRNAs in murine biofluids.
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.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, OX1 3QX Oxford, UK. robertst@scripps.edu.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Roberts
authors.forename: Anna M L
authors.initials: AM
authors.lastname: Coenen-Stass
authors.forename: Corinne A
authors.initials: CA
authors.lastname: Betts
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood