Mar 19, 2014

Detection and quantification of extracellular microRNAs in murine biofluids

Biological Procedures Online
Thomas C RobertsMatthew J A Wood

Abstract

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. 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. Here we describe robust and novel procedures to harvest mu...Continue Reading

  • References49
  • Citations8

Citations

Mentioned in this Paper

Real-Time Polymerase Chain Reaction
Ethanol
DMD gene
Phenol
Heparin
Ethanol Measurement
Whole Blood
Extracellular
Pellet Formation
RNA, Small Interfering

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