Detection and quantification of extracellular microRNAs in murine biofluids

Biological Procedures Online

Thomas C Roberts²  Anna M L Coenen-Stass¹  Corinne A Betts¹  Matthew J A Wood¹ 

[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

关键词: Plasma;    Serum;    RT-qPCR;    Biofluid;    miRNA;    Extracellular microRNA;   

Others  :  792926
DOI  :  10.1186/1480-9222-16-5

 received in 2014-02-20, accepted in 2014-02-22,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Roberts et al.; licensee BioMed Central Ltd.

【 图 表 】

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