The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality.
To overcome these deficiencies, researchers at University Medical Center Hamburg-Eppendorf developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared their assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. This new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.
Schema of reverse transcription followed by real-time PCR with
the original and designed TaqMan probe for miR-16 and miR-142
The core (stem and loop) sequences of the stem-loop primer are the same for miR-16 and miR-142, but the 3’ sticky end sequence is specific for miR-16 as published by Applied Biosystems. The forward primer (green), probe (yellow) and reverse primer (grey) were published by Chen et al. We extended the probe at its 5’ end (blue) (A). The 3’ sticky end sequence was modified to be complementary to miR-142. The forward primer sequence was modified to be specific to the miR-142 cDNA sequence. The reverse primer sequence (grey) was published by Applied Biosystems. The probe of miR-142 with the stem sequence (as published by Applied Biosystems, yellow) was extended at its 5’ end and modified at its 3’ end to create the complementarity to the miR-142 sequence (B). All modifications are in blue.