Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
The Majority of Micrornas in Serum and Saliva Is Concentrated in Exosomes.
Gallo1, Alessia, Tandon1, Mayank, Illei2, Gabor G., Alevizos1, Ilias
MicroRNAs (miRNA) are small regulatory RNA molecules with important roles in a variety of physiological and pathological processes. miRNA signatures from both unfractionated human fluids, like serum and saliva and from exosomes showed promise as diagnostic biomarkers, but the relative contribution of exosomal miRNAs to whole serum microRNAs has not yet been explored.
We first optimized a protocol to isolate exosomes from a very small amount of fresh and frozen serum and saliva. The isolation of microvescicles was done using different centrifugation and ultra centrifugation steps. The purity of exosome preparations were confirmed by electron microscopy and by Western blot using exosome specific antibodies (CD63, TSG101). The expression of selected microRNAs isolated either from exosomes or the exosome-free supernatant were compared by TaqMan RT-qPCR. We tested miR-150, miR-146a, miR-574, miR101, miR107, miR16, miR678 and miR13 in serum samples and miR202, miR203, miR1273d, and miR22 in saliva samples.
The centrifugation and ultracentrifugation steps allowed to have a very clean isolation of exosomes and subsequently a higher quality miRNA preparation, compared to the whole serum and the supernatant. The expression of all tested miRNAs in the pellet was at least 8 fold higher compared to the supernatant. The difference ranged from 8128 folds (37 cycles). Most miRNAs were either undetectable (Cycles Threshold >35) or detectable at a very low level in the exosome-free supernatant.
These results show that the majority of miRNAs in the serum and saliva is packaged within microvesicles, primarily exosomes and not found free circulating in human samples. Even though, the exosome isolation could mean an extra-step, it is maybe necessary to reliably detect miRNAs which are present at low concentration in human fluids.
To cite this abstract, please use the following information:
Gallo, Alessia, Tandon, Mayank, Illei, Gabor G., Alevizos, Ilias; The Majority of Micrornas in Serum and Saliva Is Concentrated in Exosomes. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :497